Proteomics Research 2013

Transcription

1 Proteomics Research 2013 A. Phos-tag - Phosphorylated Protein Analysis 3 1. Phos-tag Acrylamide SuperSep Phos-tag (Precast Phos-tag Acrylamide Gels) Phos-tag Biotin Phos-tag Mass Analytical Kit Phos-tag Agarose... 5 B. Protein Electrophoresis 6 1. SuperSep - Precast Polyacrylamide Gels EasySeparator Reagents for Gel Preparation Protein Size Markers Premixed Buffers Reducing Agents Staining Reagents a. Negative Gel Stain MS Kit b. Silver Staining Kits c. CBB R-250 Stain Kits - Quick CBB C. Western Blotting Membranes for Blotting Applications Blocking Reagents Wash Solutions Labeled 2 nd Antibodies Loading Control Antibodies Antibody Detection Reagent Accelerator of Antigen-Antibody Reactions Stripping Reagent Coloring Reagents Chemiluminescence Reagents D. Mass Spectrometry Reagents for Sample Pretreatment Matrices for MALDI-TOF MS In-gel Digestion Enzymes - Lysyl Endopeptidase LC/MS Solvents Related Reagents Peptide Calibration Standards of MALDI-MS... 24

2 Alphabetical Index page Description 23 A Acetic Acid 23 Acetone 23 Acetonitrile 8 Acrylamide, 99.0+% (cgc) 8 30 w/v% Acrylamide Solution, 37.5 : 1 24 ACTH (Human, 1-24) 24 Adrenomedullin (Human, 22-52) 14 Albumin, from Bovine Serum, Globulin Free 8 2-Amino-2-Hydroxymethyl-1,3-propanediol (Tris) 8 2-Amino-2-Hydroxymethyl-1,3-propanediol Amino-2-Hydroxymethyl-1,3-propanediol Hydrochloride (Tris-HCl) 23 Ammonium Hydrogencarbonate 8 Ammonium Peroxodisulfate (APS) 8 10 w/v% Ammonium Peroxodisulfate Solution (10 w/v% APS) 24 Angiotensin II (Human) 15 Anti Mouse IgG (H+L), Goat, IgG Whole, Biotin Conjugated, affinity purified Anti Mouse IgG (H+L), Rabbit, IgG Whole, Peroxidase Conjugated Anti Rabbit IgG (H+L), Goat, IgG Whole, Biotin Conjugated, affinity purified Anti Rabbit IgG (Fc), Mouse, Peroxidase Conjugated 15 Anti β-actin, Monoclonal Antibody Anti β-actin, Monoclonal Antibody, Peroxidase Conjugated 24 Apamin 17 B BCIP/NBT Solution 14 C ClearTrans Nitrocellulose Membrane, 0.2μm ClearTrans Nylon Membrane, 0.45μm ClearTrans SP PVDF Membrane, Hydrophobic, 0.2μm 21 α-cyano-4-hydroxycinnamic Acid (CHCA) 21 D 2,5-Dihydroxybenzoic Acid (DHB) 2,5-Dihydroxybenzoic Acid Lithium Salt 2,5-Dihydroxybenzoic Acid Sodium Salt 9 (+/-)-Dithiothreitol 7 E EasySeparator 22 Endoproteinase Asp-N, Sequencing grade Endoproteinase Glu-C, Sequencing grade 23 F Fluorescamine 23 Formic Acid v% Formic Acid-Acetonitrile 14 G Gelatin, from Bovine Bone 8 Glycine 23 H Hydrochloric Acid 20 Hydrophobic Protein Preparation Reagent 16 I Immuno-enhancer Immuno-enhancer Reagent A Immuno-enhancer Reagent B 18, 19 ImmunoStar LD 19 ImmunoStar Reagents 19 ImmunoStar Zeta 24 Insulin (Human) 23 Iodoacetamide 23 Iodoacetic Acid 22 L Lysyl Endopeptidase, Mass Spectrometry Grade 9 M 3-Mercapto-1,2-propanediol 9 2-Mercaptoethanol 23 Methanol 8 N,N'-Methylenebis (acrylamide)-hg 15, 16 Multi Capture-HRP 10 N Negative Gel Stain MS Kit 24 P PAMP (Rat) 9 1 PBS(-) 9 1 PBS(-)-T PBS(-) (ph 7.4) <Nippon Gene> 14 PBS-T, ph 7.4 ( 10) 3, 5 Phos-tag Acrylamide <Nard> 5 Phos-tag Acrylamide 5mM Aqueouws Solution <Nard> 3, 5 Phos-tag Agarose <MANAC Incorporated> 3, 5 Phos-tag Biotin BTL-104 <Nard> Phos-tag Biotin BTL-105 <Nard> Phos-tag Biotin BTL-111 1mM Aqueous Solution <Nard> 3, 5 Phos-tag Mass Analytical Kit <Nard> 23 Phthalaldehyde (o-phthalaldehyde) 17 POD Immunostain Set 17 Ponceau 3R Ponceau-3R Stain Solution page Description 23 P Potassium Hydroxide 23 2-Propanol 20 Proteome Preparation Reagent 13 Q Quick-CBB Quick-CBB Plus 8 R Riboflavin, % (Absorptiometry) 9 Running Buffer Solution ( 10) 9 S Sample Buffer Solution (2ME ) ( 2) Sample Buffer Solution (2ME ) ( 4) Sample Buffer Solution (2ME+) ( 2) Sample Buffer Solution (2ME+) ( 4) Sample Buffer Solution with 3-Mercapto-1,2-propanediol ( 2) Sample Buffer Solution with 3-Mercapto-1,2-propanediol ( 4) 8 10% SDS Solution <Nippon Gene> 9 SDS-PAGE 10 Running Buffer <Nippon Gene> 9 SDS-PAGE Buffer, ph8.5 8 Separating Gel Buffer Solution ( 4) 11 Silver Stain II Kit Wako 11 Silver Stain Kit Wako 10 Silver Stain MS Kit 21 Sinapic Acid, [SA] 14 Skim Blocker 14 Skim Milk Powder 23 Sodium Azide 23 Sodium Carbonate 8 Sodium Dodecyl Sulfate (SDS) 23 Sodium Thiosulfate 8 Stacking Gel Buffer Solution ( 4) 17 Stripping Solution 24 [D-Arg 1,D-Pro 2,D-Trp 7,9,Leu 11 ]-Substance P 7 SuperSep Ace, 6 %, 13 well SuperSep Ace, 7.5 %, 13 well, 17 well SuperSep Ace, 10 %, 13 well, 17 well SuperSep Ace, 12.5 %, 13 well, 17 well SuperSep Ace, 15 %, 13 well, 17 well SuperSep Ace, 5-12 %, 13 well, 17 well SuperSep Ace, 5-20 %, 13 well, 17 well SuperSep Ace, %, 13 well, 17 well SuperSep Ace, %, 13 well (Tricine Gel) 7 SuperSep, 12.5 %, 2D SuperSep, 5-20 %, 2D SuperSep, %, 2D 4 SuperSep Phos-tag (50 μmol/l), 10 %, 13 well, 17 well SuperSep Phos-tag (50 μmol/l), 12.5 %, 13 well, 17 well SuperSep Phos-tag (50 μmol/l), 15 %, 13 well, 17 well SuperSep Phos-tag (50 μmol/l), 17.5 %, 13 well, 17 well 9 T 1 TAE 9 1 TBS 9 1 TBS-T 14 TBS-T, ph 7.4 ( 10) TBS ( ph 7.4) <Nippon Gene> TBS ( ph 7.4) <Nippon Gene> mol/l TCEP Solution, Neutral 9 TCEP Hydrochloride 8 N,N,N',N' -Tetramethylethylenediamine (TEMED) 23 N,N,N',N' -Tetramethylethylenediamine 23 Thiourea 17 TMB Solution (for Membrane) 8 Tricine <Dojindo> 9 Tricine Running Buffer Solution ( 10) (Tris / Tricine / SDS Buffer) 9 10 Tris-Glycine Buffer 8 Tris-HCl Buffer Powder (1mol/L, ph 8.0) 8 1M Tris-HCl ( ph 7.0) <Nippon Gene> 1M Tris-HCl ( ph 7.5) <Nippon Gene> 1M Tris-HCl ( ph 8.0) <Nippon Gene> 1M Tris-HCl ( ph 8.5) <Nippon Gene> 1M Tris-HCl ( ph 8.8) <Nippon Gene> 1M Tris-HCl ( ph 9.0) <Nippon Gene> 22 Trypsin, from Porcine Pancreas, Mass Spectrometry Grade 23 U Ultrapure Water 22 V V8 Protease [Endoproteinase Glu-C] 23 W Wakopak MS-5C18GT 8 WIDE-VIEW Prestained Protein Size Marker (15-140kDa) WIDE-VIEW Prestained Protein Size Marker III (11-245kDa)

3 Proteomics Research 2013 A. Phos-tag - Phosphorylated Protein Analysis Phos-tag Series Phos-tag is... Phos-tag is a functional molecule that binds specifically phosphorylated ions. Basic structure of Phos-tag A Phos-tag Selectivity of binding of a phosphate ion (2-) is much higher than that of other anions. A stable complex is formed under physiological conditions (ph 5 to 8*). *: In case of Zn 2+ is used as M 2+. Zinc ion or Manganese ion Description Application Phos-tag Acrylamide Specific separation of phosphorylated proteins corresponding to the degree of phosphorylation Phos-tag Biotin Specific detection without any anti-phosphorylated antibodies on western blot Phos-tag Agarose Enrichment, separation and purification of phorphorylated proteins using column chromatography Phos-tag Mass Analytical Kit Analysis: Applicable to MALDI-TOF/Mass analysis of phosphorylated compounds in positive mode using an isotopic Zn 2+ -Phos-tag complex Phos-tag was developed by Department of Functional Molecular Science at Hiroshima University. 1. Phos-tag Acrylamide (Please see the product list on the page #5.) Separation of phosphorylated protein isoforms using SDS-PAGE Phos-tag Acrylamide is added when a resolving gel of SDS-PAGE is prepared. Since Phos-tag in the gel can trap phosphorylated proteins and the migration speed of phosphorylated proteins decreases, they can be separated from non-phosphorylated proteins. (Phos-tag SDS-PAGE). Recognition of all phosphorylated forms of Ser / Thr / Tyr Applicable to Western blotting and Mass analysis Simultaneous detection of phosphorylated- and nonphosphorylated proteins using the total antibody without the anti phosphorylated protein. Two metal ions work together with Phos-tag capture phosphate group Isoforms with different numbers and positions of phosphorylation can also be separated. Application Time course of phosphorylation by using the Tyrosine Kinase Abl Phosphorylated proteins migrate with be trapped by Phos-tag in the gel SDS-PAGE GEL Phos-tag Phosphorylated protein Non-phosphorylated protein Since the migration speed of phosphorylated proteins is decreased, they can be separated from non-phosphorylated proteins. Conventional SDS-PAGE (one band was detected.) Phos-tag SDS-PAGE (several bands were detected.) Phosphorylated tyrosine was prepared by GST binding protein of tyrosine kinase Abl and the substrate peptide (Abltide) and separated with conventional SDS-PAGE and Phos-tag SDS-PAGE, respectively. Kinase Reaction Time 0 Principle Conventional SDS-PAGE (CBB staining) (min) Kinase Reaction Time Phosphorylated proteins Phos-tag SDS-PAGE (min) Phosphorylated and Non-phosphorylated proteins Inseparable Non-phosphorylated proteins Separable For the first trial of Phos-Tag Acrylamide, the ready-to-use 5mM aqueous solution is available. (Please see the product list on the page #5.) Phosphorylated proteins + non-phosphorylated proteins 3

4 Phos-tag A 2. SuperSep Phos-tag - Precast Phos-tag Acrylamide Gels This is a precast gel added with Phos-tag Acrylamide in advance. It can be used immediately after opening the package. It contains zinc as a supplemental metal. It has excellent storage stability by its neutral gel buffer. Sharp bands can be obtained. Phosphorylated- and non-phosphorylated proteins are efficiently separable with sharp bands. Long-term stability (Stable for 6 months) Physical Properties Plate size (mm) Gel size (mm) Well number Phos-tag conc. 50 μmol/l Acrylamide conc. 10%, 12.5%, 15% or 17.5% ZnCl 2 conc. 100 μmol/l Well volume 30 μl 25 μl Application Separation of phosphoprotein! SuperSep Phos-tag SuperSep SuperSep Phos-tag (Wako's precast gel) + p.6 Application 2 M M [Buffer for electrophoresis] Tris-Glycine-SDS electrophoresis buffer [Sample for electrophoresis] 1 : α-casein 2 : Dephosphorylated α-casein [Condition of electrophoresis] Constant current 40 ma for 35 min. [Staining] Quick CBB Staining [Destaining] Destaining with methanol-acetic acid decoloring agent for 30 min. α-casein was processed with alkaline phosphatase and subjected to electophoresis using SuperSep Phos-tag and 12.5% SuperSep as a control. Band shift by dephosphorylation was observed. Phosphorylation/dephosphorylation can be quantified! [Buffer for electrophoresis] Tris-Glycine-SDS electrophoresis buffer [Sample for electrophoresis] M: WIDE-VIEW Prestained Protein Size Marker III * 1: 0 min. Albumin (no AP treatment) 2: 15 min. Albumin (AP treatment) 3: 30 min. β-casein (AP treatment) 4: 45 min. β-casein (AP treatment) 5: 60 min. β-casein (AP treatment) [Condition of electrophoresis] Constant current 35 ma for 60 min. [Staining] Quick-CBB Staining [Destaining] Deionized water with microwave β-casein was dephosphorylated over time. Bands of dephosphorylated β-casein increased over time. SuperSep Phos-tag SuperSep Description Application Wako Cat. No. Package Size SuperSep Phos-tag (50 μmol/l), 10%, 13 well sheets SuperSep Phos-tag (50 μmol/l), 10%, 17 well sheets SuperSep Phos-tag (50 μmol/l), 12.5 %, 13 well sheets SuperSep Phos-tag (50 μmol/l), 12.5 %, 17 well Separation of phosphorylated and non-phosphorylated sheets SuperSep Phos-tag (50 μmol/l), 15 %, 13 well proteins by SDS-PAGE sheets SuperSep Phos-tag (50 μmol/l), 15 %, 17 well sheets SuperSep Phos-tag (50 μmol/l), 17.5%, 13 well sheets SuperSep Phos-tag (50 μmol/l), 17.5%, 17 well sheets *: Note Prestain marker does not reflect the molecular weight, when a gel containing Phos-tag is used. Please use the result as an index of transfer efficiency at Western blotting. WIDE-VIEW Prestained Protein Size Marker III (See the page #8.) is used in this marker. At Western blotting, Zn 2+ must be sufficiently removed with transfer buffer containing 10 mm EDTA. Please wash out the gel 10 min 3 times. Subsequently, perform the same procedure 10 min. 1 using transfer buffer without EDTA, then transfer the gel. 4

5 Proteomics Research Phos-tag Biotin Detection of phosphoproteins for Western blotting This is biotin-bound Phos-tag used for detection of phosphoprotein by Western blotting. Western Blot Analysis with Phos-tag Biotin A Phos-tag All phosphoproteins can be detected. Procedures of experiment are similar to those in ordinary Western blotting. It can be conveniently used even when target anti-phosphorylated Thr/ Ser antibody is not available! BTL-104 and BTL-105 have linkers with different length, but their usage are similar. BTL-104 is recommendable as the first choice because of its high solubility. 4. Phos-tag Mass Analytical Kit Improves Detection sensitivity of MALDI-TOF MS Before use, Phos-tag Mass Analytical Kit is mixed with samples for MALDI-TOF MS. Phosphorylated molecule-phos-tag complex is detected in a positive mode, and phosphorylated molecule usually difficult to detect can be detected with improved sensitivity. Detection sensitivity of phosphorylated molecule is improved. PVDF membrane Phosphorylated protein Kit Contents w Phos-tag MS-101L 5 mg 1 vial ([C 27 H 29 N 6 O 64 Zn 2 ] 3+ MW : 581.4) w Phos-tag MS-101H 5 mg 1 vial ([C 27 H 29 N 6 O 68 Zn 2 ] 3+ MW : 589.4) w Phos-tag MS-101N 10 mg 1 vial (C 27 H 29 N 6 OZn 2 ] 3+ MW : 584.3) 101L, 101H and 101N have different types of Zinc. 101N contains many isotopes because it uses natural Zinc that has complex mass spectrum. It is suited for evaluation of condition. 101L and 101H contain Zinc with mass numbers of 64 and 68, respectively. They are suited for detection of phosphate group. 5. Phos-tag Agarose Purification of phosphorylated protein by affinity chromatography Fill Phos-tag Agarose in a column for use. It can be used for separation, purification and concentration of phosphorylated proteins. Because it is free from a surfactant or reducing agents, phosphorylated protein can be obtained in a condition similar to in vivo one. M M Phosphorylated proteins can be purified in 1 hour. The proteins can be trapped in physiological condition (ph 7.5). No reducing agent or surfactant is necessary. Description (Manufacturer) Grade Phos-tag Acrylamide (Nard) Phos-tag Acrylamide 5 mm Aqueous Soln. (Nard) Separation of phosphorylated / non-phosphorylated proteins by SDS-PAGE Application Purification of phosphorylated protein in A431 lysate M: Molecular Weight Marker Lane1: Non absorbed fraction Lane2: Absorbed fraction Lane3: Column rinsing fraction (left) Fluorescence staining (right) Western blotting with Anti-phosphorylated Tyr. Lysate was applied on a column filled with Phos-tag Agarose. Manufacturer's product No. Wako Cat. No. Package Size AAL-107M mg AAL mg AAL-107S ml Phos-tag Biotin BTL-104 (Nard) They can be used in place of anti-phosphorylated BTL mg Phos-tag Biotin BTL-105 (Nard) antibody. Any amino acids can be detected. BTL mg Phos-tag Biotin BTL-111 1mM Aqueous Solution (Nard) BTL-111 offers higher sensitivity than BTL-104. BTL-111S ml Phos-tag Mass Analytical Kit (Nard) MALDI-TOF MS MS-101KIT set Phos-tag Agarose (Manac) Purification of phosphorylated proteins AG ml AG ml 5

6 B. Protein Electrophoresis 1. SuperSep - Precast Polyacrylamide Gels SuperSep is a precast polyacrylamide gel for electrophoresis of proteins or nucleic acids. Because the gel does not contain SDS, it can be used for SDS-PAGE in SDS-containing buffer, and for Native-PAGE in buffers that do not contain SDS. 12 kinds of precast gels are available. Protein Electrophoresis B Example of Electrophoresis [SuperSep Ace 15-20% (Tricine Gel)] [Application] [Gel] SuperSep Ace 15-20%, 13well (Tricine Gel) [# ] [Running Buffer] Tricine Running Buffer Soln.( 10) [# ] [Condition of electrophoresis] Constant current 20mA [Staining] Quick-CBB PLUS [# ] [Lane2] Molecular Weight Marker, Low Range [Lane4] Molecular Weight Marker, Middle Range [Lane6] Molecular Weight Marker, Wide Range [Note] When using tricine gels, we recommend using the tricine buffer ( 10) (# , Composition: 0.5 M tris/0.5 M tricine/1% SDS) as an electrophoresis buffer to obtain more accurate molecular weight than using a tris-glycine buffer. Product Specification Well Well volume (μl) Mass of Total Protein (μg)* 3.3 ~ ~ 3.9 Plate Size (mm) Gel Size (mm) *Target amount of protein that can be separated efficiently with SuperSep gel Note: In order to obtain sharp bands, apply a sample as little as possible slowly to the bottom of each well. When much sample is applied, resultant bands may be blurred. It is advisable to rinse inside of each well with a syringe or a fine pipette, before applying samples. Tips - How to remove a gel from a glass plate 1. Easily-breakable method 2. Break-proof method If you insert a spatula between a spacer and a gel and pull the gel off horizontally, you may make a small cut on the gel. Even such a minor cut may make the gel easily tearable. Insert a spatula vertically into a gel and push the gel off the glass plate. This method does not damage the gel and makes the gel more untearable in the subsequent operation. Target of separation Please select appropriate gel concentration, with reference to the following separation pattern. 6

7 Proteomics Research 2013 Description well Package Size Wako Cat. No. Shelf life SuperSep Ace, 6% sheets months SuperSep Ace, 7.5% sheets sheets SuperSep Ace, 10% sheets sheets SuperSep Ace, 12.5% sheets sheets months SuperSep Ace, 15% sheets sheets SuperSep Ace, 5-12% sheets sheets months SuperSep Ace, 5-20% sheets sheets SuperSep Ace, 10-20% sheets sheets months SuperSep Ace, 15-20% (Tricine Gel) sheets months SuperSep, 12.5%, 2D 2D 10 sheets SuperSep, 5-20%, 2D 2D 10 sheets months SuperSep, 10-20%, 2D 2D 10 sheets B Protein Electrophoresis *Shelf life after manufacturing date 2. EasySeparator This is an electrophoresis tank for SuperSep precast polyacrylamide gel. Attachment and removal of the gel are easy. Turn the knob after electrophoresis, then running buffer filled inside in the tank easily falls down. So, the gel can be removed directly. Easy set-up of SuperSep by turning the knob. Easy removal of SuperSep without disposing the inside running buffer after electrophoresis. Electrophoresis can be performed with a minimum amount of running buffer (250 ~ 350 ml) Easy Setting Easy Removing 1. Insert the gel. 2. Turn the knob. 3. Setting of the gel is complete! 1. Remove the cover. 2. Turn the knob. (The inside running buffer falls down.) 3. Remove the gel itself without disposing the buffer. Description Wako Cat. No. Package Size EasySeparator set 7

8 3. Reagents for Gel Preparation Followings are reagents used to prepare polyacrylamide gel for protein electrophoresis. Various kinds of reagents such as powder and ready-to-use solution are available. Protein Electrophoresis B Description Grade* Wako Cat. No. (Package Size) Acrylamide, 99.0+% (cgc) (25 g); (100 g); (500 g) 30 w/v% Acrylamide Solution, 37.5 : (500 ml) Separating Gel Buffer Solution ( 4) (0.4 w/v% SDS in 1.5mol/L Tris-HCl, ph 8.8) for Electrophoresis (250 ml) Stacking Gel Buffer Solution ( 4), (0.4 w/v% SDS in 1.5mol/L Tris-HCl, ph 6.8) (250 ml) N,N'-Methylenebis (acrylamide) -HG, 99.0+% (Titration) (1 g); (25 g); (100 g) N,N'-Methylenebis (acrylamide) -HG, 99.0+% (Titration) for Molecular Biology (25 g); (100 g) N,N,N',N'-Tetramethylethylenediamine (TEMED), 99.0+% (cgc) (25 ml) Ammonium Peroxodisulfate (APS), 99+ % (Titration) 10 w/v% Ammonium Peroxodisulfate Soln. (10 w/v% APS) for Electrophoresis (10 g); (100 g) (25 ml) Riboflavin, % (Absorptiometry) (1 g) 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris), % (Titration) 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris), % (Titration) 2-Amino-2-hydroxymethyl-1,3-propanediol 999, % (Titration) Sodium Dodecyl Sulfate (SDS), % (GC) ; % (Titration) for Molecular Biology for Biochemistry (100 g); (500 g); (1 kg) (100 g); (500 g); (2.5 kg) (100 g); (500 g); (1 kg); (5 kg) (25 g); (100 g); (500 g) Sodium Dodecyl Sulfate (SDS), % for Molecular Biology (25 g); (100 g); (500 g) 10% SDS Solution Nippon Gene (100 ml); (500 ml) 2-Amino-2-Hydroxymethyl-1,3-propanediol Hydrochloride (Tris-HCl) 2-Amino-2-hydroxymethyl-1,3-propanediol Hydrochloride (Tris-HCl) for Biochemistry for Molecular Biology (500 g) Tris-HCl Buffer Powder (1 mol/l, ph 8.0) for Genetic Research (1 L 4) 1M Tris-HCl (ph 7.0) 1M Tris-HCl (ph 7.5) 1M Tris-HCl (ph 8.0) 1M Tris-HCl (ph 8.5) 1M Tris-HCl (ph 8.8) 1M Tris-HCl (ph 9.0) Nippon Gene (100 g); (500 g); (1 kg) (100 ml); (500 ml) (100 ml); (500 ml) (100 ml); (500 ml) (100 ml); (500 ml) (100 ml); (500 ml) (100 ml); (500 ml) Glycine 99.0+% (Titration) (25 g); (100 g); (500 g) Tricine Dojindo (25 g); (100 g) * : for Molecular Biology - A series of reagents with DNase and RNase activities checked. 4. Protein Size Markers Recombinant protein markers bound to stains in advance. It is useful for confirming progress of electrophoresis or transfer efficiency. No. j k WIDE-VIEW Prestained Protein Size Marker III (11 ~ 245 kda) WIDE-VIEW Prestained Protein Size Marker (15 ~ 140 kda) for Electrophoresis μl (for 100 tests) 500 μl (for 50 ~ 100 tests) (kda) red j Prestained III green (kda) pink k Prestained 8

9 Proteomics Research Premixed Buffers Reducing Agents This is a series of ready-to-use premixed buffers for protein electrophoresis. They can be used immediately after dilution. Running Buffers for SDS-PAGE Description Package Application Wako Cat. No. Composition Size Running Buffer Solution ( 10) (Tris / Glycine / SDS Buffer) Laemmli method Tris-Glycine Composition: 250 mmol/l Tris, 1,920 mmol/l Glycine, 1.0% (w/v) SDS Running Buffer containing SDS L SDS-PAGE 10 Running Buffer <Nippon Gene> Laemmli method Tris-Glycine L Composition: 250 mmol/l Tris, 1,920 mmol/l Glycine, 1.0% (w/v) SDS Running Buffer containing SDS L 10 Tris-Glycine Buffer Composition: 0.25 mol/l Tris 1.92 mol/l Glycin Native-PAGE, Transfer Buffer L Tricine Running Buffer Solution ( 10) (Tris / Tricine / SDS Buffer) Composition: 0.5 M Tris / 0.5 M Tricine /1% SDS Running Buffer for Tris-Tricine gel L Electrophoresis Sample Buffers Description Composition Sample Buffer Solution (2-ME+) ( 2) Composition: mol/l Tris-HCl (ph 6.8), 4w/v% SDS, 20 w/v% Glycerol, w/v% BPB, 10 vol% 2-Mercaptoethanol Sample Buffer Solution (2-ME+) ( 4) Composition: 0.25 mol/l Tris-HCl (ph 6.8), 8 w/v% SDS, 40 w/v% Glycerol, 0.02 w/v% BPB, 20 vol% 2-Mercaptoethanol Sample Buffer Solution (2-ME-) ( 2) Composition: mol/l Tris-HCl (ph 6.8), 4w/v% SDS, 20 w/v% Glycerol, w/v% BPB Sample Buffer Solution (2-ME-) ( 4) Composition: 0.25 mol/l Tris-HCl (ph 6.8), 8 w/v% SDS, 40 w/v% Glycerol, 0.02 w/v% BPB Sample Buffer Solution with 3-Mercapto-1,2-propandiol ( 2) Composition: mol/l Tris-HCl (ph 6.8), 4 w/v% SDS, 20 w/v% Glycerol, 0.01 w/v% BPB, 10 v/v% 3-mercapto-1,2-propandiol Sample Buffer Solution with 3-Mercapto-1,2-propanediol ( 4) Composition: 0.25 mol/l Tris-HCl (ph 6.8), 8 w/v% SDS, 40 w/v% Glycerol, 0.02 w/v% BPB, 20 v/v% 3-mercapto-1,2-propandiol Application Laemmli Sample Buffer with 2-Mercaptoethanol Laemmli Sample Buffer without 2-Mercaptoethanol Laemmli Sample Buffer containing 3-Mercapto-1,2- propanediol (non-hazardous chemical) as substitute for 2-ME Wako Cat. No. Package Size ml ml ml ml ml ml B Protein Electrophoresis 1 Buffer Series The followings do not require dilution and are ready to use. The buffers are offered in sterile 5 L containers with capped stopcocks. 1 PBS(-) L 1 PBS(-)-T L for Biochemistry 1 TBS L 1 TBS-T L SDS-PAGE Buffer, ph 8.5 for Electrophoresis L 1 TAE for Genetic Research L Reducing Agents This is a water-soluble reductant that cleaves disulfide bonds in proteins ml 5A 2-Mercaptoethanol for Biochemistry g g g g (+/-)-Dithiothreitol SH Groups Protection g g mg 3-Mercapto-1,2-propanediol 98.0+% (Titration) ml ml g TCEP Hydrochloride g for Biochemistry ml 0.5 mol/l TCEP Solution, Neutral ml 9

10 6. Staining Reagents a. Negative Gel Stain MS Kit Protein Electrophoresis B It is known that protein bands separated by SDS/polyacrylamide gel electrophoresis (SDS-PAGE) can be visualized as transparent bands against the background of milky white gel stained by negative gel stain containing a Zn/imidazol reagent. We have improved the method using a new imidazol derivative reagent, which allows a clear and stable image of protein bands on the gel as sensitive as that by silver staining, in as little as 10 minutes. The staining technique is useful to obtain the clear and sensitive resolution pattern of the gel before immunoblotting as well as to excise and purify the band of interest from the gel without significant deterioration of amino acid residues for the subsequent studies of protein such as sequencing and mass analysis of peptide. Staining Principle Staining uses insoluble opaque ZnIm 2 generated by reaction of free Zn 2+ and imidazole. Protein and protein-sds complex react with Zn 2+. The moiety reacted with Zn 2+ remains clear because insoluble ZnIm 2 does not deposit on it, but the moiety other than protein becomes opaque because ZnIm 2 deposits on it. This type of staining is called negative staining, because unlike other staining, background is stained and protein is not stained. High sensitive (3 ng) and Quick detection (10 minutes). Applicable to the subsequent studies of protein such as sequencing and mass analysis of peptide. Repeatable staining and destaining are acceptable. After destaining, the gel is applicable to Western blotting. MALDI-TOF MS of rat phosphorylase excising MS measurement The obtained band was excised and in-gel digested with trypsin. Then the sample was analyzed on MALDI-TOF mass spectrometer. (These data were provided by Dr. Y. Wada at Osaka Medical Center, Japan.) Kit Contents w Staining Solution A w Staining Solution B w De-staining Solution 500 ml 1 bottle 500 ml 1 bottle 500 ml 1 bottle Negative Gel Stain MS Kit for Electrophoresis tests b. Silver Staining Kits 3 types of high sensitive Silver Staining Kits are available. Principle Silver ion selectively binds to sulfhydryl group (-SH), amino group (-NH 2 ), hydroxyl group (-OH), or carboxyl group (-COOH), which consist of protein. Especially, reaction with sulfhydryl group (-SH) occurs preferentially. Silver ion forming complex with ammonium ion in the reagent binds to protein, via substitution of ammonium ion and sulfhydryl group in protein. Silver Stain MS Kit This kit is produced for mass spectrometric sequencing of proteins separated by polyacrylamide gel electrophoresis, based on the method described by Shevchenko, et al. Due to the omission of glutaraldehyde treatment, proteins are rarely modified chemically and therefore can be detected at a sub-nanogram level on the electrophoretic gel. Kit Contents w Enhancing Stock Soln. 200 ml 1 w Stopper 200 ml 1 w Staining Stock Soln. 200 ml 1 w De-staining Soln. A 50 ml 1 w Developing Stock Soln. 100 ml 1 w De-staining Soln. B 50 ml 1 w Developing Powder 20 g 1 Application MALDI-TOF MS of rabbit phosphorylase The band was excised and treated with Lysyl Endopeptidase (# ). Following the in-gel digestion and preparation, the sample was analyzed on MALDI-TOF mass spectrometer. (These data were provided by Dr. Y. Wada at Osaka Medical Center, Japan.) Staining: Silver Stain MS Kit (Wako) rabbit phosphorylase, In-gel digest with lysylendopeptidase 10 Silver Stain MS Kit for Electrophoresis tests

11 Proteomics Research 2013 Silver Stain Kit Wako Silver Staining Kit Wako has high sensitivity and reproducibility. Staining finishes in 100 minutes. Kit Contents w Fixing Stock Soln. (Glutaraldehyde) 200 ml 2 w Enhancing Stock Soln (Dithiothreitol) 10 ml 1 w Staining Soln. A (Silver nitrate) 100 ml 1 w Staining Soln. B (Ammonia & Sodium Hydroxide) 100 ml 1 w Developing Stock Soln. 100 ml 1 (Formaldehyde & Citric Acid) Gel: SuperSep 15%, 12 well Sample: Protein Size Marker B Silver Stain Kit Wako for Electrophoresis for 10 sheets Silver Stain II Kit Wako An improved type Silver Staining kit which can stain protein bands in 1 hour. It contains Stopper, which can be used for adjustment of staining for your intended use. Protein Electrophoresis Kit Contents (100 ml each 1 bottle) w Fixing Stock Soln. (Methanol & Acetic Acid) w Enhancing Stock Soln. (Dithiothreitol & Glutaraldehyde) w Staining Soln. A (Silver Nitrate) w Staining Soln. B (Ammonia & Sodium Hydroxide) w Developing Stock Soln. (Formaldehyde & Citric Acid) w Stopper (Citric Acid) Gel: SuperSep 12.5%, 12 well Sample: Rough extract of E. coli. Silver Stain II Kit Wako for Electrophoresis for 10 sheets Please Visit Wako's Online Catalog: 50,000 products for Life Science, Green Chemistry, Analytical Chemistry! Flexible search function: Chemical Name Wako Catalog No. Molecular Formula CAS Number... 11

12 Wako's Silver Staining Kits Protein Electrophoresis B Procedure Reagent Wako Cat. # Silver Stain Kit Wako supplies II MS (Wako Cat. # ) (Wako Cat. # ) Negative Gel Stain MS Kit (Wako Cat. # ) Glutaraldehyde Containing Containing Free Free Gel after electrophosing with SDS-Polyacrylamide gel or Polyacrylamide gel 1. Fixing-1 Fixing Soln min. (Note 1) 10 min. (Note 1) 20 min. (Note 1) Skip (Note 1) 2. Fixing-2 Fixing Soln min. 10 min. 10 min. (Note 1) 3. Wash (Deionized Water) 5 min. 3 times 10 min. 4. Enahncing Enhancing Soln. 5 ~10 min. 10 min. 1 min. 5. Wash (Deionized Water) 5 min. 5 min. 1 min. 2 times 6. Silver Staining Staining Soln. 15 min. 15 min. 20 min. Staining Soln A 5~10 min. 7. Wash (Deionized Water) 3~5 min. 3 times 2~5 min. 3 times 1 min. 2 times Wash 5 ~10 sec. 3 times 8. Development Developing Soln. 5 min. 5 min. 3 ~10 min. Staining Soln. B 10~60 sec. 9. Stopping Stopper 2~3 min. (Note 1) 2~3 min. 1 min. 10. Wash (Deionized water) 2 min. 3 times 2 min. 3 times 1 min. 3 times Wash 1 min. 3 times (abt. 90 min.) (abt. 70 min.) (abt. 70 min.) (abt. 10 min.) Separation of gel (separation of target protein) (Note 2) (Note 2) 11. Destaining 15 min. 5 min. (Note 3) In-gel Digestion (Note 4) MS analysis Fixing Solution Fixing Stock Soln. Fixing Stock Soln. 200 ml ml 1 Enhancing Soln. Enhancing Stock Soln. Enhancing Stock Soln. Enhancing Stock Soln. 10 ml ml ml 1 Kit Contents (Note 5) Staining Soln. Developing Soln. Staining Soln. A 100 ml 1 Staining Soln. B 100 ml 1 Developing Stock Soln. 100 ml 1 Staining Soln. A 100 ml 1 Staining Soln. B 100 ml 1 Developing Stock Soln. 100 ml 1 Staining Stock Soln. 200 ml 1 Developing Stock Soln. 100 ml 1 Developing Powder 20 g 1 Stopper Stopper Stopper 100 ml ml 1 De-staining Soln. A De-staining Soln. 50 ml 1 De-staining Soln. B 50 ml 1 Staining Soln. A 500 ml 1 Staining Soln. B 500 ml 1 Destaining Soln. 500 ml 1 (Note 1) Since these kits do not contain this reagent, please prepare it by yourself. When you use Negative Gel Stain MS Kit, it is not necessary to fix samples. However, higher sensitivity will be obtained with 50 % ethanol fixing for 5 minutes (Note 2) Brown colored staining parts are excised in case of silver staining. On the other hand, black colored parts against a black paper are excised in case of negative gel staining. (Note 3) When used in subsequent MS analysis, de-staining is unnecessary. When used is subsequent Western Blotting, the gel is de-stained. (Note 4) Lysyl Endopeptidase, Mass Spectrometry Grade (Wako Cat. No ; 20 μg 5) / Trypsin, from Porcine Pancreas, Mass Spectrometry Grade (Wako Cat. # ; 20 μg 5) are available from Wako. (Please see the page #22.) (Note 5) Each Silver Stain Kit does not contain deionized water, methanol and acetic acid. Please prepare by yourself. (The above mentioned reagents are not necessary when you use Negative Gel Stain MS Kit.) 12

13 Proteomics Research 2013 c. CBB R-250 Stain Kits Quick CBB Quick CBB is an upgraded version of the conventional CBB stain using CBB R-250. After fixing treatment, proteins are stained by immersing the gel in Quick CBB solution. Protein bands can be detected in a short time after SDS-PAGE treatment because no destaining procedure with acetic acid is necessary. Detectable in 50 minutes. Easy preparation. (Prepare Staining Soln. by mixing Soln. A and B at a ratio of 1 : 1) 10 minute detection with microwave. (Application 2) Gel: SuperSep 5-20%, 12 well Sample: Rough extract of E. coli. Protein Size Marker Description Kit Content Grade Wako Cat. No. Package Size Quick CBB jstaining Solution A (1 L 1) kstaining Solution B (1 L 1) for Electrophoresis L Quick-CBB PLUS This is a protein staining kit which is a further upgraded version of Quick-CBB. In comparison with conventional Quick- CBB, fixing procedure is not required and organic solvents such as methanol and acetic acid are not used. The mixing procedure is unnecessary as all the required solutions for staining are contained in a single bottle. There are other improvements, such as the removal of background coloration. As in conventional Quick-CBB, destaining procedure is not required. B Protein Electrophoresis No fixing procedure and no organic solvents are necessary. A single-bottled ready-to-use protein staining kit Simple and quick staining without the coloration on background. Quick-CBB PLUS ml for Electrophoresis L Application 1 j k l m n o p q r s Regular Protocol Wash: Deionized water 100 ml (5 min. 3 times) Staining: Quick-CBB PLUS 25 ml (30 ~ 60 min.) Wash: Deionized water 100 ml Gel: SuparSep Ace 5-20%, 17 well Sample: j~n 5, 4, 3, 2, 1 μl (in that order)/ WIDE-VIEW Prestained Protein Size Marker (# ) o~s 5, 4, 3, 2, 1 μl (in that order)of Molecular Weight Marker, Wide Range (# ) Application 2 10 minute protocol by microwave With Quick-CBB PLUS, staining and decoloring can be completed in about 10 min using microwave. It is very useful when users want the result at once. [Protocol] 1. SDS-PAGE 2. Staining by microwave: for 1 min. 2 ~ 4 times (In case of Quick-CBB, for 1 min 1 time) Pour Quick-CBB PLUS, cover with plastic wrap and heat at 500 W for 1 minute. Repeat microwaving till the whole gel get colored. 3. Destaining by microwave: 1 min. 4 ~ 6 times Discard Quick-CBB PLUS from the tray and pour 100 ml of deionized water to immerse the gel. Add a balled-up Kimwipe in the tray and heat for a few minutes. Repeat this destaining procedure with new deionized water.* * In case of bumping, there is no problem if the gel is immersed in the solution. Leave the gel in deionized water overnight to lower the background. (Caution) When the tray is completely covered with plastic wrap, make several holes on the wrap with needle. The tray becomes very hot. Use gloves and pay full attention when removing the tray. Ordinary staining j k l m n o p q r [Result of staining] Staining by microwave j k l m n o p q r Sample: BSA Sample Amount: j Total Protein weight: 5 μg k~r are 2-fold dilution series of j Electrophoresis: SuperSep 15%, 12 well 13

14 C. Western Blotting 1. Membranes for Blotting Applications ClearTrans Membrane Series ClearTrans SP PVDF Membrane, Hydrophobic This PVDF membrane can be used for fluorescent protein detection. It is used for highly sensitive detection of proteins due to its strong affinity with proteins. It also provides for a high S/N ratio because of the low background noise levels. It has small pores with diameters of 0.2 μm that reduce the permeability of proteins with low molecular weight. ClearTrans Nitrocellulose Membrane This product is Nitrocellulose Membrane for transcription. It can be used for colony/plaque lifts as well as nucleic acid blots. [Dimensions] 30 cm 3 m, [Pore size] 0.2 μm ClearTrans Nitrocellulose Membrane This non-charged membrane for blotting applications is made of nylon 6-6. It can be used for colony/plaque lifts as well as nucleic acid blots. Western Blotting C ClearTrans SP PVDF Membrane, Hydrophobic, 0.2 μm roll (26 cm 3 m) ClearTrans Nitrocellulose Membrane, 0.2 μm for Blotting roll (30 cm 3 m) ClearTrans Nylon Membrane, 0.45 μm roll (30 cm 3 m) 2. Blocking Reagents Blocking reagents used to prevent non-specific absorption of antibody to the transfer membrane. Generally, Bovine Serum Alubumin (BSA) or Skim Milk are used as a blocking reagent. Skim Milk is a potent blocking reagent, but it is not suitable for phosphorylated antibodies because they contain a lot of phosphorylated proteins. Skim Blocker Skim Blocker is a blocking reagent that prevents non-specific absorption to the membrane or ELISA plates. This is a readyto-use reagent that can be used immediately. Blocking finishes by shaking for 30 minutes at room temperature. Skim Blocker ml for Blotting Skim Milk Powder g Gelatin, from Bovine Bone* g g for Biochemistry g Albumin, from Bovine Serum, Globulin Free* g g *: Not available for sale in the US. 3. Wash Solutions Wash solution is a buffer for washing membranes for Western blotting or ELISA plates. It can be easily prepared by diluting the following solution with distilled water. Description Composition PBS-T, ph7.4 ( 10) Composition: 1,370 mmol/l NaCl, 81 mmol/l Na 2 HPO 4, 27 mmol/l KCl, 15 mmol/l KH 2 PO 4, 1 (w/v)% Tween 20 TBS-T, ph7.4 ( 10) Composition: 1,370 mmol/l NaCl, 27 mmol/l KCl, 250 mmol/l Tris, 1 (w/v)% Tween TBS (ph7.4) Composition: 1,370 mmol/l NaCl, 26.8 mmol/l KCl, 250 mmol/l Tris 20 TBS (ph7.4) Composition: 400 mm Tris-HCl (ph 7.4), 3 M NaCl 10 PBS ( ) (ph7.4) Composition: 1,370 mmol/l NaCl, 81 mmol/l Na 2 HPO 4, 26.8 mmol/l KCl, 14.7 mmol/l KH 2 PO 4 Grade Wako Cat. No. Package Size L for Blotting L ml Nippon Gene ml ml 14

15 Proteomics Research Labeled 2 nd Antibodies Various types of labeled secondary antibodies are available. Anti-mouse IgG, HRP labeled antibody, anti-rabbit IgG and HRP labeled antibodies are now available. Application 1 Application 2 Wako Company G Sample: mixture of FLAG-BAP and THP-1 lysate Primary antibody: Anti FLAG, MAb / 2 nd antibody: Anti Mouse IgG (H+L), rabbit, IgG Whole, POD conjugated ( ) Lane 1: 5,000 Lane 2: 10,000 Lane 3: 20,000 Lane 4: 40,000 Wako Company G Sample: mixture of FLAG-BAP and FM3A cell lysate Primary Antibody: Anti FLAG, Polyclonal Ab 2 nd antibody: Anti Rabbit IgG (Fc), mouse, POD conjugated (# ) Lane 1: 2,500 Lane 2: 5,000 Lane 3: 10,000 Lane 4: 20,000 Description 5. Loading Control Antibodies The followings are a monoclonal antibody for β-actin. A protein expressed by many eukaryotic cells, β-actin is widely used as a loading control in Western blotting μg Anti β-actin, Monoclonal Antibody mg for Immunochemistry mg Anti β-actin, Monoclonal Antibody, Peroxidase Conjugated μl Grade Wako Catalog No. Package Size Anti Mouse IgG (H+L), Rabbit, IgG Whole, Peroxidase Conjugated μl ml Anti Rabbit IgG (Fc), Mouse, Peroxidase Conjugated for Immunochemistry μl ml Anti Mouse IgG (H+L), Goat, IgG Whole, Biotin Conjugated, affinity purified mg Anti Rabbit IgG (H+L), Goat, IgG Whole, Biotin Conjugated, affinity purified mg C Western Blotting 6. Antibody Detection Reagent Multi Capture HRP This is a reagent kit binding to Fc region of antibodies to detect antibodies. Chemiluminescence can be used for detection because the capture reagent is HRP-labeled. Mouse IgG 1 may be difficult to detect. In that case, use the Enhancer Reagent (for mouse) provided in the kit. Almost all antibodies can be detected. (Mouse IgG 1 and Goat IgG may be difficult to detect.) Sensitivity is similar or higher than that existing secondary antibodies. Low lot-to-lot variation chemiluminescent reagent membrane Antigen primary antibody Capture Reagent peroxidase A molecule of Capture Reagent is labeled with approx. 50 of peroxidase. Application 1 High Sensitivitiy Application 2 Enhancement effect to mouse antibody Primary Ab: Anti FLAG, rabbit, polyclonal Ab (0.5 μg/ml) 2 nd Antibody: 1: Multi Capture HRP ( 5,000) 2: Multi Capture HRP ( 10,000) 3: Multi Capture HRP ( 20,000) 4: HRP conjugated Anti rabbit IgG, Polyclonal Ab ( 10,000) Primary Ab: Anti FLAG, mouse IgG 1, MAb (1 μg/ml) 2 nd Antibody: 1: Multi Capture HRP ( 2,000) 2: Multi Capture HRP ( 2,000) + Enhancer Reagent for Mouse 3: Anti Mouse IgG (H+L), Rabbit, IgG Whole, POD Conjugated ( 5,000) (Wako Cat. # ) 15

16 Kit Contents 10 tests* (Wako Cat. # ) 40 tests* (Wako Cat. # ) (1) Capture Reagent 50 μl 200 μl (2) 20 Reaction Buffer 10 ml 40 ml (3) Enhancer Reagent for Mouse 50 μl 200 μl *The package size is based on the use when capture reagent is diluted to 1 : 2,000. Multi Capture HRP tests for Blotting tests 7. Accelerator of Antigen-Antibody Reactions Western Blotting C This product is an accelerator to optimize antigen-antibody reaction for Western blotting, dot blotting, enzyme-linked immunosorbent assay (ELISA). Immuno-enhancer aids in obtaining high signal-to-noise (S/N) ratio. Enhances Immunoassay signals, effectively High S/N Ratio Applicable to Ready-to-Use Antibody Diluent Kit contents for 2 assays* for 10 assays* for 40 assays* Reagent A 10 ml 50 ml 200 ml Reagent B 10 ml 50 ml 200 ml *Each package size shows an assay number when 5 ml each is used per assay. Application 1 Application 2 Application Procedure 1/1 1/2 1/4 1/8 1/1 1/2 1/4 1/8 Western blotting SDS-PAGE Transfer to a membrane Blocking Reaction with Primary Antibody Reaction with 2 nd Antibody Detection Note: In case of one-step antigen-antibody reaction with a labeled antibody, dilute the antibody with reagent B. Atg Diluted with Reagent A Diluted with Reagent B Antibodies: Primary Antibody: Anti Rat LC3 antiserum, rabbit Secondary Antibody: Anti rabbit- IgG-HRP antibody Exposure time: 15 seconds Immunoenhancer Skim-milk A549 cell Lysate 5 g ( 1) or 10 g ( 2) were subjected to SDS-PAGE and blotted onto nitrocellulose membranes. They were blocked and Western blot. 3% skim milk in TBS-T solution was used as the control. Antibodies: Primary Antibody: Anti EB1, Rabbit (1:500), 2 hour-reaction Secondary Antibody: HRP conjugated Anti rabbit IgG (1 : 7000), 1 hour-reaction Exposure time: 10 seconds Immuno-enhancer TBS-T HeLa Cell Lysate, diluted by 1/1, 1/2, 1/4 and 1/8 were subjected to SDS-PAGE and blotted onto a membrane, blocked, followed by Western blotting. TBS-T solution was used as the control. Antibodies: Primary Ab: Anti Actin, Goat (0.5 g/ml), 1 hour-reaction Secondary Ab: Anti goat-igg-hrp (1:10,000 dilution), 1 hour-reaction Exposure time: 5 minutes Immunoenhancer 1% BSA LC3-I LC3-II Cell extract was separated by 12.5% SDS-PAGE, transferred onto PVDF membrane, followed by Western blotting the primary antibody diluted with Immuno-enhancer Reagent A. 20 mm Tris-HCl (ph 7.5)-0.15 M NaCl-0.1% NaN 3 containing 1% BSA was used as the control. The secondary antibody was diluted with 20 mm Tris-HCl (ph 7.5)-0.15 M NaCl-0.05% Tween 20. Generally, LC3-I form is shown in Atg- cell and LC3-II is mainly detectable in Atg7+. To detect LC3-I in Atg7+, it is necessary to lengthen the exposure time. By using Immuno-enhancer, achieve LC3-I detection, even if the shorter exposure time. (Data was provided by Dr. Takashi Ueno, Dep. of Biochemistry, Juntendo Univ. School of Medicine.) assays Immuno-enhancer assays for Blotting assays Immuno-enhancer Reagent A ml Immuno-enhancer Reagent B ml 16

17 Proteomics Research Stripping Reagent Stripping Solution is used to remove primary and secondary antibodies from a Western Blot membrane. Stripping is useful when several proteins are investigated on a sheet of membrane. Usage Wash a membrane reacted with a labeled antibody using TBS-T or PBS-T, and shake the membrane with Stripping Solution for 10 min. at RT. Saving your time & samples Only one electrophoresis Antibodies can be removed in 10 min. Application Sample: Mixture of 1 and 2 1. Transthyretin Variant (L55P), Human, recombinant (Wako Cat. # ) 2. Glutathione S-Transferase Membrane: PVDF Membrane; Blocking: 0.5% BSA Labeled Antibody: Anti 6x His, MAb (9C11), HRP conjugated (Wako Cat.# ) 4 ml Chemiluminescent reagent: ImmunoStar LD (Wako Cat. # ) Exposure time: 1 second Wash the membrane with TBS-T Incubate the membrane on an orbital shaker with Stripping Solution for 10 minutes Wash the membrane with TBS-T Blocking: 0.5% BSA Labeled Ab: Anti Glutathione S-transferase, MAb, Peroxidase Conjugated Chemiluminescent reagent: ImmunoStar LD (# ); Exposure time: 1 second Stripping Solution for Blotting ml 9. Coloring Reagents TMB Solution (for Membrane) Protein 1 Stripping treatment 10 minutes Protein 2 Protein 1 After detection of Protein 1, the antibody was removed with Stripping Solution, and subsequently Protein 2 was detected using the antibody. TMB (3,3',5,5'-tetramethylbenzidine) reacts with peroxidase to generate a bluish purple precipitate. This enables one-step detection of HRP-tagged antigens on membranes after Western blots, without the need for any special equipment. C Western Blotting L TMB Solution (for Membrane) for Blotting ml POD Immunostain Set This is a sensitive enzyme-immunostaining kit of horseradish peroxidaselabeled immunoglobulins on nitrocellulose membrane. Phenol is oxidized by reaction of phenol and hydrogen peroxide in the coloring reagent with POD, and blue-purple diformazan is generated in proportion to POD activity, in the presence of NADH and NTB. Antigen is detected by this reaction. Kit Contents w NTB solution w NADH w Substrate solution 250 ml 1 bottle for 20 ml 12 bottles 13 ml 1 bottle POD Immunostain Set for POD Staining ml 12 BCIP/NBT Solution BCIP (5-Bromo-4-chloro-3-indolyl-phosphate) and NBT (Nitroblue Tetrazolium) generate an insoluble, dark blue-purple colored product by alkaline phosphatase. This reaction can be used for detection by Western blotting. The solution is prepared as a substrate for coloring in advance at a concentration suitable for immunoblotting. This product is inapplicable to a microwell-eia and immunohistostaining. BCIP / NBT Solution for Biochemistry ml Ponceau 3R The product is used for staining serum proteins by cellulose acetate membrane electrophoresis g Ponceau 3R for Electrophoresis g Ponceau-3R Stain Solution ml 17

18 10. Chemiluminescence Reagents ImmunoStar LD High-sensitive chemiluminescent detection in a short time ImmunoStar LD (Long Detection), which is designed for a simple and highly sensitive immunoblotting, utilizing detection by enhanced chemiluminescence using our unique luminol derivative L-012 as the substrate can be detect labeled HRP. Signal of Western blotting can be detected with high sensitivity due to combination with unique enhancer. Also, it has long duration of luminescence. Principle L-012 is a luminol derivative, and dianion generated from H 2 O 2 shows chemiluminescence. Western Blotting C N NH 2 Cl L-012 O O N N H 2 O 2 +HRP N NH 2 Cl High Sensitivity (10-14 ; femto gram level) Long term duration of chemiluminescence (CHL) (24 hours) Low background (a high ratio of S/N) Easy preparation (Prepare Luminescence Working Solution by mixing Soln. A and B at a ratio of 1:1) High-sensitive detection in a short time A good result with low background can be obtained by exposure for several seconds to minutes. O O O O excited-state NH 2 OH + N 2 + Chemiluminescence N OH (λmax 458nm) Cl O O ground-state Aβ37 Aβ38 Aβ39 Aβ40 Aβ42 1 (50pg) ImmunoStar LD 1/2 (25pg) 1/4 (12.5pg) 1/8 (6.25pg) 1/16 (3.12pg) 1/32 (1.5pg) High sensitivity product (Company T) 1 (50pg) 1/2 (25pg) 1/4 (12.5pg) 1/8 (6.25pg) 1/16 (3.12pg) 1/32 (1.5pg) Exposure time 15 sec. A mixture of synthesized Aβ, 37, 38, 39, 40 and 42 was subjected to SDS-PAGE using Tris-Tricine Urea gel that can separate Aβ molecule species, then transferred to PVDF membrane and boiled up. Western blotting was performed using an antibody specific to N terminal of human Aβ. 30 sec. 1 min. Detection: LAS-1000plus (Data was provided by Dr. Tomita and Osawa, Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo) References 1. Obana, N., et al. : Mol. Microbiol., 77, 1416 (2010) 2. Fukumoto, H., et al. : J. Neurosci., 30, (2010) 3. Hyakkoku, K., et al. : Neuroscience., 171, 258 (2010) 4. Obana, N., et al. : J. Bacteriol., 193, 4417 (2011) 5. Kurotani, R., et al. : J. Biol. Chem., 286, (2011) 18

19 Proteomics Research 2013 ImmunoStar Zeta Long-lasting stable luminescence (mid-femtogram: g) ImmunoStar Zeta using L-012, a luminol derivative, as a substrate. It can keep background noise low and detect chemiluminescence with a high S/N ratio. It also shows stable luminescence signals for a long period of time. A reagent that exhibits long-lasting stable luminescence Low background (high S/N ratio) 2 component (equal proportions) type [Electrophoretic Condition] [Gel] SuperSep Ace, % (Wako Cat. # ) [Sample] DYKDDDDK-BAP [Lane1] 20 ng, [Lane2] 10 ng, [Lane3] 5 ng, [Lane4] 2.5 ng, [Lane5] 1.25 ng [Antibody Response Condition] [Blocking ] 5% Skim-milk PBST 1hr [Primary Ab] Anti DYKDDDDK tag, Monoclonal Antibody (Wako Cat. # )(5,000 dilution), 1 hour-reaction [Secondary Ab] Anti Mouse IgG(H+L)-HPR (Wako Cat. # )(20,000 dilution), Over night-reaction C Western Blotting Showed more stable luminescence stability than competitors products did. ImmunoStar Reagents A colorimetric method and UV detections are available after luminescence detection (picogram: g) The ImmunoStar Reagents is a luminescent reagent whose detection limits for protein are in the picogram range. A color and UV detection are available after luminescence detection. For UV detection, irradiate UV light after leaving the luminescent membrane for one night. A color and UV detection are available after luminescence detection. Low background (high S/N ratio) 3 component type Description Characteristics Detection Limit Kit components Grade Wako Cat. No. Package Size Useful where high cm 2 sensitivity is required, low femtogram 2 components ImmunoStar LD* such as in detecting g (equal proportions) underexpressed proteins ,000 cm 2 2,000 cm 2 ImmunoStar Zeta* A reagent that exhibits long-lasting stable luminescence mid femtogram g 2 components (equal proportions) for Blotting cm ,000 cm ,000 cm 2 ImmunoStar Reagents A colorimetric method and UV detections are available after luminescence detection picogram g 3 components ,000 cm ,000 cm 2 *: Not available for sale in the US and Europe. 19

20 D. Mass Spectrometry Gel Proteomics Add high-purity Matrix CHCA (# ) SA (# ) DHB (# ) See page #21 Sample Pretreatment (See page #20) Protein identification Analysis of posttranslational protein modification Sampling Electrophoresis Negative Gel Stain MS Kit (# ) See page #10 Silver Stain MS Kit (# ) See page #10 In-gel digestion Lysyl Endopeptidase, MS Grade (# ) Trypsin, from Porcine Pancreas, MS Grade (# ) See page #22 Mass spectrometry by MALDI-TOF MS 1. Reagents for Sample Pretreatment Proteome Preparation Reagent Mass Spectrometry D This product easily removes mucopolysaccharides and nucleic acids from extracted samples of plants, animals and microorganisms. As a result, the viscosity is decreased and the figure of electrophoresis is improved. Usage (1) Add Proteome Preparation Reagent to each sample. (1/20 volume of the sample) (2) Mix and leave it at 4 C. (3) Centrifuge at 12,000 rpm at 4 C for 5 min. (4) Collect the supernatant (5) Electrophorese Purpose of use: for elimination of slime polysaccharides contained in plant extract liquid and nucleic acids in biological extract samples. Proteome Preparation Reagent for Proteome Research μl Hydrophobic Protein Preparation Reagent Fig. 1 Pretreatment Effect of yam aerial tuber extract Pretreatment with Proteome Without pretreatment Preparation Reagent Fig. 2 Pretreatment Effect of chicken cardiomyocyte extract Pretreatment with Proteome Without pretreatment Preparation Reagent (Data was provided by T. Araki at Laboratory of Biochemistry, School of Agriculture, Kyushu Tokai University) This product is used to accelerate solubilization of hydrophobic proteins and to obtain two-dimensional electrophoretic spots of proteins that cannot be isolated by a conventional method. It is also used to reduce the effects of lipids and nucleic acids contaminated in soluble proteins and obtain clearer spots. Usage 1 (1) Prepare target protein fraction (2) Suspend in Lysis Buffer. Purpose of use: for solubilization of insoluble proteins such as a membrane protein contained in extracts of microorganisms or animal tissues. (3) Add Hydrophobic Protein Preparation Reagent (1/3 volume of the sample) (4) Mix and incubate at 30 C for 30 min. (5) Centrifuge at 14,000 rpm for 15 min. (6) Collect the supernatant. Usage 2 (1) Appropriately prepare protein for 2D electrophoresis Purpose of use: for reducing the effects of lipids or nucleic acids contaminated in soluble proteins. (2) Add 1/5 volume of Hydrophobic Protein Preparation Reagent into the prepared sample solution. (3) Mix and leave at RT/30 C for 30 min. (4) Collect the supernatant ml Hydrophobic Protein Preparation Reagent for Proteome Research ml

21 Proteomics Research Matrices for MALDI-TOF MS High-purity Matrix for MALDI-TOF MS CHCA, SA and DHB Add High-purity Matrix, such as Wako s CHCA, SA or DHB Sample Pretreatment Protein identification Analysis of posttranslational protein modification Sampling Electrophoresis In-gel digestion Mass spectrometry 1. High purity matrix is mixed with a test sample and used for proteome analysis by mass spectrometer (MALDI-TOF MS). 2. High purity matrix gives good mass spec reading because of its high purity by recrystallization). Effects of recrystallization of α-cyano-4-hydroxycinnamic Acid (CHCA) When comparing with MALDI-TOF MS data for commercial CHCA, it was found that recrystallized CHCA (Wako Cat. No ) gives clearer mass spec readings with less background noise. (These data were provided by Dr. Wada Y. at Osaka Medical Center, Japan) Untreated CHCA Recrystallized CHCA D % intensity % intensity Mass Spectrometry Mass (m/z) Mass (m/z) Untreated CHCA Recrystallized CHCA with the peptide sample Product List α-cyano-4-hydroxycinnamic Acid [CHCA] mg 5 Sinapic Acid [SA] mg 5 2,5-Dihydroxybenzoic Acid [DHB] for Proteome Research mg 5 2,5-Dihydroxybenzoic Acid Lithium Salt mg 2,5-Dihydroxybenzoic Acid Sodium Salt mg 21

22 3. In-gel Digestion Enzymes Lysyl Endopeptidase, MS Grade / Trypsin, from Porcine Pancreas, MS Grade Among the most important techniques in proteome analyses is the in-gel digestion of protein spots / bands that have been resolved by electrophoresis using digestive enzymes, such as trypsin and lysyl endopoptidase. Proteins can be identified by mass spectrometry of the peptides produced by in-gel digestion, and further information regarding post-translational modifications can be obtained. Lysyl Endopeptidase, Mass Spectrometry Grade is a lyophilized product that retains sufficient activity for in-gel digestion and packed in very small quantities for convenience purposes. 1. High specificity and efficiency of protein digestion allow for easy database searches by peptide mass. 2. Improved cleavage at lysine residue and increase in the number of peptides are obtained by combination with trypsin. 3. Packed in very small quantities in accordance with common usage guidelines, so sufficient activity is retained for in-gel digestion. Comparison of In-gel Digestion Using Trypsin (Tp), Lysyl Endopeptidase (Lep) and Lep Combined with Tp (Lep +Tp) BSA band (100 ng) resolved by SDS-PAGE was in-gel digested with Tp, Lep and Lep +Tp and analyzed by MALDI-TOF MS. The figure shows the individual mass spectra. The evaluation of these peptidases is summarized in the table. Table: Comparison of Tp, Lep and Lep +Tp These results indicate there are very few missed cleavages obtained by Lep digestion. When Tp is used concomitantly with Lep, missed cleavages decrease and the number of identified peptides increase compared to when only Tp is used. Tp Lep Lep +Tp Cleavage site C terminal of Arg and Lys C terminal of Lys C terminal of Arg and Lys Missed cleavage (Rates of missed cleavage)*1 Many (8 %) Very few (0 %) Few (3 %) No. of identified peptides *1 The value resulted from subtracting the coverage obtained when database searches were performed with Missed cleavage 0 from that obtained when performed with Missed cleavage 1. "Coverage" is the percentage of peptides obtained after in-gel digestion in the whole sequence. Mass Spectrometry D a Tp b Improved coverage Lep + Tp Figure: Comparison of the mass spectra of (a) Trypsin (Tp) and (b) Lysyl Endopeptidase (Lep) +Tp The peaks at m/z 2000 were obtained after digestion with Lep + Tp, but not Tp alone. These results indicate improved sequence coverage. (Data provided by Dr. Y. Wada, Osaka Medical Center and Research Institute for Maternal and Child Health) Lysyl Endopeptidase, Mass Spectrometry Grade μg 5 for Proteome Research Trypsin, from Porcine Pancreas, Mass Spectrometry Grade* μg 5 *2 : Not available for sale in the US Related Products Endoproteinase Asp-N, Sequencing grade μg for Sequencing Grade Endoproteinase Glu-C, Sequencing grade μg V8 Protease [Endoproteinase Glu-C] for Biochemistry mg References 1. Wada, Y., and Kadoya, M.: J. Mass Spectrom., 38, 117 (2003). 2. Shevchenko, A., Wilm, MM., Vorm, O., and Mann., M.: Anal. Chem., 68, 850 (1996). 22

23 Proteomics Research LC/MS Solvents Liquid chromatography - mass spectrometry (LC/MS) is widely used in various fields including biological, food, and environmental analyses. In particular, recent breakthroughs in the development and upgrades of device interfaces have led to the use of LC/MS in microanalyses of environmental pollutants and chemical metabolites, etc. Following products are ideal LC/MS reagent to analyze trace components. Description Wako Cat. No. Package Size Specification Acetic Acid Acetonitrile Formic Acid (abt. 99%) 0.1 vol% Formic Acid-Acetonitrile Methanol 2-Propanol Ultrapure Water Related Products ml 5 A ml. Suitability test for LC/MS performed. Reduced background noise ml. Suitability test for LC/MS performed. Guarantees noise level at m/z 50~2, L. Use of aluminum caps Reduced risks of slight amounts of contaminants L from plastic caps ml 5 A ml. Suitability test for LC/MS performed. Reduced background noise L. Suitability test for LC/MS analysis performed L Ready-to-Use eluent ml. Suitability test for LC/MS performed. Guarantees noise level at m/z 50~2, L. Use of aluminum caps Reduced risks of slight amounts of contaminants L from plastic caps L L. Suitability test for LC/MS performed L. Decreased total organic carbon levels. Guarantees the absorbance and fluorescence tests. Use of specially processed glass containers / L aluminum caps Assay (HPLC): 99.5+% Absorbance (1 4,250 nm): max Absorbance (1 4,254 nm): max Fluorescence test: to pass test Suitability for LC/MS analysis: to pass test Assay (cgc): 99.8+% Density (20 C): ~ g/ml Fluorescence test: to pass test Suitability for LC/MS analysis: to pass test Assay (HPLC): 99.5+% Solubility in water: to pass test Absorbance (1 4,254 nm): max.1.00 Fluorescence test: to pass test Suitability for LC/MS analysis: to pass test Absorbance ( nm): to pass test Fluorescence test: to pass test Water: max. 0.05% Assay (cgc): 99.7+% Density (20 C): 0.789~0.792 g/ml Fluorescence test: to pass test Suitability for LC/MS analysis: to pass test 99.7+% (cgc) Density (20 C): 0.784~0.787 g/ml Fluorescence test: to pass test Suitability for LC/MS analysis: to pass test Density (20 C): ~ g/ml Refractive index nd20: ~ Absorbance (210~400 nm): max Fluorescence test: to pass test Total organic carbon (TOC): max. 4 ppb Description Wako Cat. No. Package Size Joint Type 2.0 mmf 50 mm. The glass lined inside wall of the stainless column allows Wakopak maximum inactivation mmf 100 mm MS-5C18GT. DuPont Excellent peak shapes and recovery rates in analyses of trace 2.0 mmf 150 mm components within biological samples 5. Related Reagents D Mass Spectrometry Acetone ml ml Ammonium Hydrogencarbonate g g Fluorescamine mg Hydrochloric Acid ml ml Iodoacetamide g g Iodoacetic Acid g g Phthalaldehyde (o-phthalaldehyde) g for Proteomics g Potassium Hydroxide g g Sodium Azide g g Sodium Carbonate g g Sodium Thiosulfate g g N,N,N',N'-Tetramethylethylenediamine ml ml Thiourea g g 23

24 6. Peptide Calibration Standards of MALDI-MS For MALDI-MS Calibration Standard Peptide standards for matrix-assisted laser desorption ionization (MALDI) mass spectrometry used extensively for analysis of biological polymers such as proteins are highly purified meeting the requirements of MALDI-MS. They are available in step sizes of 500 in molecular weight for selection of calibrants corresponding to your sample. 1. High Purity 2. Appropriate calibrant for your sample 3. Cost-conscious 2 nmol package [MS Chart Example 1] Example of Use µlh2o % Intensity In MALDI-MS assay, clear MS spectrum with a very small amount of metal additives is obtained. H2O or 0.1% TFA 20 µl 1µL 0.5 µl 0.5 µl Mass Spectrometry D Sample: Angiotensin II (Wako Cat. No ) Matrix: CHCA (Wako Cat. No ) [MS Chart Example 2] [MS Chart Example 3] % Intensity ( ) ( ) Mass (m/z) ( ) ( ) ( ) Mass (m/z) ( ) ( ) % Intensity Sample: Insulin Stock Solution Working Solution Sample: MS Calibrant mixture Matrix: CHCA (Wako Cat. No ) (*: Peptide with M. W is under consideration for commercialization.) (Data were provided by WADA Yoshinao, Osaka Medical Center & Research Institute for Maternal & Child Health) Description (M+H) + Grade Wako Cat. No. Package Size Angiotensin II (Human) nmol 1 bottle j <MALDI-MS Calibrant> nmol 5 bottles [D-Arg 1, D-Pro 2, D-Trp 7,9, Leu 11 ]-Substance P nmol 1 bottle k <MALDI-MS Calibrant> nmol 5 bottles Apamin nmol 1 bottle l <MALDI-MS Calibrant> nmol 5 bottles PAMP (Rat) nmol 1 bottle m for Proteome Research <MALDI-MS Calibrant> nmol 5 bottles ACTH (Human, 1-24) nmol 1 bottle n <MALDI-MS Calibrant> nmol 5 bottles Adrenomedullin (Human, 22-52) nmol 1 bottle o <MALDI-MS Calibrant> nmol 5 bottles Insulin (Human) nmol 1 bottle p <MALDI-MS Calibrant> nmol 5 bottles ( ) Mass (m/z) MALDI target Matrix Solution Listed products are intended for laboratory research use only, and not to be used for drug, food or human use. Please visit our online catalog to search for other products from Wako ; This brochure may contain products that cannot be exported to your country due to regulations. Bulk quote requests for some products are welcomed. Please contact us. Wako Pure Chemical Industries, Ltd , Doshomachi 3-Chome Chuo-Ku, Osaka , Japan Tel: Fax: Wako Chemicals USA, Inc. Toll-Free (U.S. only): Head Office (Richmond, VA): Tel: / Fax: Los Angeles Sales Office (Irvine, CA): Tel: / Fax: Boston Sales Office (Cambridge, MA): Tel: / Fax: Wako Chemicals GmbH European Office: Fuggerstra e 12, D Neuss, Germany Tel: Fax: GK