Supplementary Figure 1. Method development.
|
|
- Theodora McCoy
- 5 years ago
- Views:
Transcription
1 Supplementary Figure 1 Method development. Titration experiments to determine standard antibody:lysate concentration. Lysates (~2 mg of total proteins) were prepared from cells expressing FLAG- tagged CBX2 (a) or SCMH1 (b) and either anti-flag or a number of recombinant antibodies were titred in for immunoprecipitation. Shown is a Western blot of the Flag-tagged antigen. The left of each Panel, labeled IP, indicates the proteins captured in the IP and on the right, labeled FT, are the proteins remaining in the lysate after immunoprecipitation. Most of the recombinant Fabs approached saturation when 2 µg of antibody or 10 µl of anti-flag beads were used per 2 mg of lysate. Nature Methods doi: /nmeth.3472
2 Supplementary Figure 2 Determination of a suitable cutoff for the elimination of background contaminants. (a) The graph shows the number of preys recovered at a given percentage of occurence among all Fab or IgG purifications. From this graph it seems that 8% is an inflection point indicating a good cut-off frequency. Below 8%, increase of percentage causes rapid drop in the number of preys recovered while above 8% the drop in frequency slows down dramatically. This implies that preys appearing in less than 8% purifications are more likely to be specific. Those appearing in more than 8% purification are more likely to be non-specific (regardless of the occurrence, the number of preys is similar). (b) Venn diagrams showing overlap between proteins identified in immunoprecipitations using >1,000 different recombinant antibodies and those from
3 Crapome repository 11. On the left, we compared all the proteins detected in all immunoprecipitations and on the right only those proteins that appear in more than 8% of all purifications.
4
5 Supplementary Figure 3 Comparison between IP-WB and IP-MS methods. Fabs or IgGs (phagemid IDs are indicated) against several targets were used to immunoprecipitate their antigens from HEK293 cell extracts expressing their respective Flag-tagged proteins. The immunoprecipitated proteins were then detected using either WB or MS. The spectral counts from IP-MS experiments are indicated below each panel.
6
7 Supplementary Figure 4 Comparison of staining patterns. Flag-tagged cell lines were stained using either anti-flag antibody or Fabs against 5 different proteins, listed on the right, and the staining pattern was compared. For these Fabs, the localization patterns are very similar to the anti-flag staining. DAPI staining was used to highlight the nuclei. Flag-tagged WDR5, shows nucleolar staining, but the anti-wdr5 Fab does not; the nucleolar staining is likely due to the over-expression of Flagtagged WDR5. Scale represents 20 µm.
8 Supplementary Protocol: IP- MS NOTE: Everything on ice or at 4 o C unless indicated otherwise. All incubations are with rotation. NOTE: Use non-autoclaved tubes and tips! A. Preparing cell lysate (HEK293 cells) 1. Thaw frozen cell pellets immediately with 1X High Salt AFC buffer (1 ml per 15 cm plate) with protease and phosphatase inhibitors 2. Perform 3 freeze- thaw cycles by moving samples between ethanol/dry ice and 37 C water bath, mixing frequently to prevent temperature from reaching above 4 C 3. Sonicate, (Branson sonicator) 5x (0.3s on/0.7 s off) per 1 ml, 8x per 2 ml and 12x per 5 ml 4. Incubate for 30min at 4 C with benzonase nuclease to remove RNA and DNA (to the final concentration of units/ml). 5. Centrifuge at 13,000 rpm for 30min at 4 C. Discard pellet and retain supernatant. 6. Measure protein concentration (nanodrop), usually 7-10 mg/ml B. Antibody binding 1. Use 2-5 mg of total protein per IP. Add ~2 μg of antibody into the lysate and incubate at 4 o C overnight (depending on the stability of the proteins), 2 hrs incubation is sufficient for most antibodies. 2. Wash 20 µl of magnetic beads per IP twice with 100 µl high salt AFC buffer. After last wash resuspend in 20 µl of low salt AFC buffer and add 20 µl to the lysates with antibodies. 3. Incubate for 2-4 hrs at 4 o C. 4. Wash the beads with 1 ml of 1x low salt lysis buffer 3x at 4 o C for 5-10 min and 2x with low salt lysis buffer without detergent. 5. Elute 4x50 µl of 0.5 M ammonium hydroxide, combine the elutions and flash freeze in liquid nitrogen 6. Dry and perform trypsin digest according to protocol below. C. Trypsin Digestion NOTE: All the steps should be done in HPLC grade water or 50mM NH4HCO3 NOTE: Use non- autoclaved tubes and tips NOTE: Make sure you work in a clean environment to prevent keratins and polymer contamination, hood is not necessary but careful work environment is essential! 1. Dry NH4OH samples in the speed vac (about hrs) 2. Reconstitute in 44 µl of 50 mm NH4HCO3 3. Add 1 µl of 100mM TCEP- HCL. Shake at 37 o C for 1 hr
9 4. Cool to room temperature and add 1 µl 500 mm iodoacetamide. Shake at room temperature and in dark for 45 mins 5. Add 1 µg Trypsin. Shake overnight at 37 o C 6. Add 2µl acetic acid to stop the reaction 7. Store at 4 o C 8. The volume is 50.5 µl D. Desalting (for Zip- Tip) NOTE: The maximum volume for Zip Tip is 10 µl 1. Equilibration: Aspirate 10µl Wetting and Equilibration solution into zip- tip. Dispense waste. Repeat twice. Aspirate 10µl with washing solution. Dispense waste. Repeat twice. 2. Binding and washing: Aspirate 10υl and dispense into the sample, repeat at least 20 times so all the peptides are bound to the tip (do not throw away the samples just pipette up and down). Aspirate 10υl of washing solution twice and dispense. 3. Elution: Dispense 10υl of Wetting and Equilibrating solution into 96 well plate four times (Final sample volume is 40 µl). 4. Dry the samples. 2. AFC (High Salt) (Lysis buffer) Final con Stock For 40 ml 10X buffer Tirs- HCl, ph7.9 10mM 2 M 2 ml NaCl 420mM 5 M 33.6 ml NP % 10% 4 ml H2O 0.4 ml Add protease inhibitor just before use Add 1mM Ni to the buffer for demethylases 3. AFC (Low Salt) (Dialysis/Wash buffer) Final con Stock For 40 ml 10X buffer Tirs- HCl, ph mm 2 M 2 ml NaCl 100 mm 5 M 8 ml NP % 10% 4 ml H2O 26 ml Add protease inhibitors just before use, same as high salt 4. Wetting and Equilibration solution 70% CAN in 0.1% FA
10 5. Washing solution 100% H20 in 0.1% FA 6.Catalogue # Zip Tip catalogue # ZTC18M960 (Millipore) Mass Spec Plate Cat no: PCR- 96- FS- C (Axygen scientific from VWR)
11 Supplementary Note Trypsin Digestion Protocols Lab 1 (Greenblatt Lab): Samples were reconstituted in 44 µl of 50 mm NH4HCO3, and 1 µl of 100mM TCEP-HCL (Thermo Fisher) was added. Samples were incubated at 37 C for 1 hr with shaking and then cooled to room temperature. 1 µl 500 mm iodoacetamide was added and samples incubated in the dark at room temperature for 45 mins, following which 1 µg of trypsin (Promega, Catalog #V5113) was added per sample and samples incubated overnight at 37 C. The next day, 2µl of acetic acid was added to stop the reaction. The final volume was 50.5µl. Desalting was performed using ZIP-TIP (Millipore, Catalog #ZTC18M960). ZIP-TIP were equilibrated by aspirating and dispensing 10 µl of wetting and equilibration solution two times followed by 2x10µl of washing solution. Binding was performed by aspirating and dispensing 10 µl of sample at least 20x to allow all the peptides to bind to the tip. The tips were then washed 2x with 10µl of washing solution, and the bound peptides eluted into a 96 well plate with 4x10µl of wetting and equilibrating solution (Axygen, Catalogue #PCR-96-FS-C). After drying and acidification to 1% formic acid, the peptides were ready to be injected into LC- MS/MS. Lab 2 (Gingras lab): Trypsin digestion was carried out on the beads. Beads with bound proteins were subjected to one rapid wash in 0.5 ml lysis buffer followed by on-bead trypsin hydrolysis for 4 h at 37 C with 0.25 µg trypsin (Sigma, Trypsin Singles). Released peptides in solution were removed and further digested overnight with an additional 0.25 µg of trypsin at 37 C. 2µl of acetic acid was added to stop the reaction. The sample was desalted using ZIP-TIP as described above. Lab 3 (Aebersold/Gstaiger): Samples were reconstituted in Resuspend in 50µl 8M urea 50 mm ammonium bicarbonate, reduced with 5 mm TCEP for 30 min and alkylated with10 mm iodoacetamide for 30 min. Samples were diluted to 300µl with 50 mm ammonium bicarbonate to reduce urea concentration and digested overnight with 1ug
12 trypsin. The next morning samples were acidified to 1.25 % with formic acid and solid phase extraction as per manufacturer using C18 ultramicrospin columns (Nest Group part SS18V) was carried out. Samples were resuspended 20 µl (0.1% formic acid 2% ACN). Lab 4 (Paulovich lab): Samples were alkylated with 50mM iodoacetamide at room temperature in the dark for 30 minutes, diluted 10 in 10 mm Tris, ph 8, and digested with Trypsin Gold (Promega, Madison, WI) at a protein-to enzyme ratio of 50:1 overnight at 37 C. The reaction was quenched by adding formic acid to a final concentration of 1%. Samples were desalted with Discovery SPERP reverse phase C18 columns(supelco) according to manufacturers protocols. Briefly, the columns were rinsed thrice with 80% acetonitrile / 0.1% formic acid (FA), equilibrated four times with 0.1% FA. The sample was applied to the column, washed 4 with 0.1% FA, and eluted with 2 washes of 50% acetonitrile / 0.1% FA, followed by 2 washes of 80% acetonitrile / 0.1% FA. The sample was concentrated by vacuum centrifugation to 20 µl. Lab 5 (Thermo): Samples were reconstituted with 50µl of 4M Urea, 300mM Tris, 2.5% n-propanol and 10mM DTT, vortexed for 10 min and incubated for 30 minutes at 37C. 4µl of 500mM IAA (iodoacetic acid) in 1M NH4HCO3 was added to the final concentration of 40 mm per well. The samples were incubated at room temperature in complete darkness for 30 min. 20 µg trypsin was with 500µl 25mM Acetic Acid (100µg vial w/2500µl AA) and incubated at 37C for 5 minutes. 75µl of 50mM Tris, 5mM CaCl2 ph 8.0 was added directly to each well followed by addition of 25ul Trypsin/AA to each well. The samples were incubated at 37C for 20 hours. Samples were quenched with 10 µl of Quench mix (20µl PRTC (Pierce Peptide Retention Time Cal Mixture 5uM), 200µl 100%Formic Acid, 16µl Glucagon [1mg/ml in H2O], 764µl LCM grade H2O.
In-Solution Digestion for proteomics
In-Solution Digestion for proteomics Guidelines for sample preparation (How to protect your samples from contamination with keratin) 1. Try to avoid any contact of samples and solutions with dust, skin
More informationChromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab)
Chromatin Immunoprecipitation (ChIPs) Protocol (Mirmira Lab) Updated 12/3/02 Reagents: ChIP sonication Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mm Tris 8.1, 150 mm NaCl, 5 mm EDTA): 10 ml 10 % Triton
More informationTrypsin Mass Spectrometry Grade
058PR-03 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Trypsin Mass Spectrometry Grade A Chemically Modified, TPCK treated, Affinity Purified
More informationAntibodies: LB1 buffer For 50 ml For 10ml For 30 ml Final 1 M HEPES, ph 2.5 ml 0.5 ml 1.5 ml 50mM. 5 M NaCl 1.4 ml 280 µl 0.
Experiment: Date: Tissue: Purpose: ChIP-Seq Antibodies: 11x cross-link buffer: Regent Stock Solution Final Vol for 10 ml of 11xstock concentration 5 M NaCl 0.1M 0.2 ml 0.5 M EDTA 1 mm 20 ul 0.5 M EGTA,
More informationMidi Plant Genomic DNA Purification Kit
Midi Plant Genomic DNA Purification Kit Cat #:DP022MD/ DP022MD-50 Size:10/50 reactions Store at RT For research use only 1 Description: The Midi Plant Genomic DNA Purification Kit provides a rapid, simple
More informationChromatin IP (Isw2) Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles.
Chromatin IP (Isw2) 7/01 Toshi last update: 06/15 Reagents Fix soln: 11% formaldehyde, 0.1 M NaCl, 1 mm EDTA, 50 mm Hepes-KOH ph 7.6. Freshly prepared. Do not store in glass bottles. 2.5 M glycine. TBS:
More informationTrypsin Digestion Mix
G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name 239PR Trypsin Digestion Mix Provides optimal buffered conditions for in gel trypsin digestion
More informationMinute TM Plasma Membrane Protein Isolation and Cell Fractionation Kit User Manual (v5)
Minute TM Plasma Membrane Protein Isolation and Cell Fractionation Kit Catalog number: SM-005 Description Minute TM plasma membrane (PM) protein isolation kit is a novel and patented native PM protein
More informationGeneaid DNA Isolation Kit
Instruction Manual Ver. 02.21.17 For Research Use Only Geneaid DNA Isolation Kit GEB100, GEB01K, GEB01K+ GEC150, GEC1.5K, GEC1.5K+ GET150, GET1.5K, GET1.5K+ GEE150, GEE1.5K, GEE1.5K+ Advantages Sample:
More informationFor the 5 GATC-overhang two-oligo adaptors set up the following reactions in 96-well plate format:
Supplementary Protocol 1. Adaptor preparation: For the 5 GATC-overhang two-oligo adaptors set up the following reactions in 96-well plate format: Per reaction X96 10X NEBuffer 2 10 µl 10 µl x 96 5 -GATC
More informationProteaseMAX Surfactant, Trypsin Enhancer
Technical Bulletin ProteaseMAX Surfactant, Trypsin Enhancer INSTRUCTIONS FOR USE OF PRODUCTS V2071 AND V2072. PRINTED IN USA. Revised 1/10 ProteaseMAX Surfactant, Trypsin Enhancer All technical literature
More informationTitle: Column Chromatography of Green Fluorescent Protein
Title: Column Chromatography of Green Fluorescent Protein Approvals: Preparer Date_07Oct06 Reviewer: Mary Jane Kurtz Date 09Jul13 Part I Crude Isolation of GFP from Lysed Cells q Page 1 of 6 1. Purpose:
More informationVirus Harvest AAV 15 cm 2
Virus Harvest AAV 15 cm 2 1) Gently scrape cells from plate in 10 ml of media, place remaining 10 ml of media in plate lid. 2) Once cells are removed from plate, wash plate by pipetting 20 ml of media
More informationAccuMAP Low ph Protein Digestion Kits
TECHNICAL MANUAL AccuMAP Low ph Protein Digestion Kits Instruc ons for Use of Products VA1040 and VA1050 5/17 TM504 AccuMAP Low ph Protein Digestion Kits All technical literature is available at: www.promega.com/protocols/
More informationTECHNICAL BULLETIN. Catalog Number RAB0447 Storage Temperature 20 C
Phospho-Stat3 (ptyr 705 ) and pan-stat3 ELISA Kit for detection of human, mouse, or rat phospho-stat3 (ptyr 705 ) and pan-stat3 in cell and tissue lysates Catalog Number RAB0447 Storage Temperature 20
More informationAMPK Assay. Require: Sigma (1L, $18.30) A4206 Aluminum foil
AMPK Assay Require: Acetone Sigma (1L, $18.30) A4206 Aluminum foil Ammonium sulfate Fisher BP212R-1 AMP Sigma A1752 ATP Sigma A6144 (alt. use A7699) Beta-mercaptoethanol Sigma M6250 (alt. use M7154) Bio-Rad
More informationProtocol for Gene Transfection & Western Blotting
The schedule and the manual of basic techniques for cell culture Advanced Protocol for Gene Transfection & Western Blotting Schedule Day 1 26/07/2008 Transfection Day 3 28/07/2008 Cell lysis Immunoprecipitation
More informationABIOpure TM Viral (version 2.0)
ABIOpure TM Viral (version 2.0) DNA/RNA Extraction Handbook Cat No: M561VT50 FOR RESEARCH USE ONLY Table of Contents Contents Page Kit Components 3 Precautions 3 Stability & Storage 4 General Description
More informationUniversal sample preparation method for proteome analysis
nature methods Universal sample preparation method for proteome analysis Jacek R Wi niewski, Alexandre Zougman, Nagarjuna Nagaraj & Matthias Mann Supplementary figures and text: Supplementary Figure 1
More informationThe Schedule and the Manual of Basic Techniques for Cell Culture
The Schedule and the Manual of Basic Techniques for Cell Culture 1 Materials Calcium Phosphate Transfection Kit: Invitrogen Cat.No.K2780-01 Falcon tube (Cat No.35-2054:12 x 75 mm, 5 ml tube) Cell: 293
More informationTrident Membrane Protein Extraction Kit
Cat. No. Size Shelf life GTX16373 5/ 20 tests 12 months at the appropriate storage temperatures (see below) Contents Component Storage Amount for 5 tests Amount for 20 tests Buffer A -20 o C 2.5 ml 10
More informationProtocol for purification of recombinant protein from 300 ml yeast culture
Protocol for purification of recombinant protein from 300 ml yeast culture Equipment and reagents needed: Zirconia beads (0.5 mm diameter from BSP, Germany) Paint Shaker (at 4 C) Tube rotator for 15 ml
More informationTECHNICAL BULLETIN. Phospho-Akt (pser 473 ) ELISA Kit for detection of human, mouse, or rat phospho-akt (pser 473 ) in cell and tissue lysates
Phospho-Akt (pser 473 ) ELISA Kit for detection of human, mouse, or rat phospho-akt (pser 473 ) in cell and tissue lysates Catalog Number RAB0011 Storage Temperature 20 C TECHNICAL BULLETIN Product Description
More informationXTRAKT FFPE Kit / Manual
XTRAKT FFPE Kit / Manual For manual extraction of RNA, mirna and/or DNA from Formalin Fixed Paraffin Embedded (FFPE) tissue samples Catalog No. # XTK2.0-96 For research use only. Not intended for diagnostic
More informationMethodology for the Extraction of Plasmodium Protein. Learning Objectives:
Proteomics Extraction of Plasmodium Protein Methodology for the Extraction of Plasmodium Protein Extraction of entire protein from the sample requires an optimized protocol and many protocols have been
More informationCell Lysis Buffer. Catalog number: AR0103
Cell Lysis Buffer Catalog number: AR0103 Boster s Cell Lysis Buffer is a ready-to-use Western blot related reagent solution used for efficient extraction of total soluble protein in nondenatured state
More informationE.Z.N.A. SQ Blood DNA Kit II. Table of Contents
E.Z.N.A. SQ Blood DNA Kit II Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Blood Storage and DNA Yield...4 Preparing Reagents...5 100-500 μl Whole Blood Protocol...6
More informationRecipes for Media and Solution Preparation SC-ura/Glucose Agar Dishes (20mL/dish, enough for 8 clones)
Protocol: 300 ml Yeast culture preparation Equipment and Reagents needed: Autoclaved toothpicks Shaker Incubator set at 30 C Incubator set at 30 C 60 mm 2 sterile petri dishes Autoclaved glass test tubes
More informationCaution: For Laboratory Use. A product for research purposes only. Eu-W1284 Iodoacetamido Chelate & Europium Standard. Product Number: AD0014
TECHNICAL DATA SHEET Lance Caution: For Laboratory Use. A product for research purposes only. Eu-W1284 Iodoacetamido Chelate & Europium Standard Product Number: AD0014 INTRODUCTION: Iodoacetamido-activated
More informationEXO-DNAc Circulating and EV-associated DNA extraction kit
Datasheet EXO-DNAc Circulating and EV-associated DNA extraction kit This product is for research use only. It is highly recommended to read this users guide in its entirety prior to using this product.
More informationEXO-DNA Circulating and EV-associated DNA extraction kit
Datasheet EXO-DNA Circulating and EV-associated DNA extraction kit This product is for research use only. It is highly recommended to read this users guide in its entirety prior to using this product.
More informationFOCUS SubCell. For the Enrichment of Subcellular Fractions. (Cat. # ) think proteins! think G-Biosciences
169PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name FOCUS SubCell For the Enrichment of Subcellular Fractions (Cat. # 786 260) think
More informationAperto Cell Lysis and Protein Solubilization Users Manual
Aperto Cell Lysis and Protein Solubilization Users Manual Revision 2 THIS MANUAL APPLIES TO THE FOLLOWING PRODUCTS: 3A8600 Aperto, 5X Cell Lysis Buffer. 20mL 3A8610 Aperto, 5X Cell Lysis Buffer. 100mL
More informationThe following protocol describes the isolation of nuclei from tissue. Item. Catalog No Manufacturer
SOP: Nuclei isolation from tissue and DNaseI treatment Date modified: 090923 Modified by: P. Sabo. (UW) The following protocol describes the isolation of nuclei from tissue. Ordering Information Item.
More informationNuclear Extraction Kit
Nuclear Extraction Kit Catalog Number KA1346 50 assays Version: 07 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Principle of the Assay... 3 General Information... 4
More informationBench Protocol to Perform TAILS v4
Bench Protocol to Perform TAILS v4 OVERALL Lab Protocols May 2016 We have been performing TAILS for 12 years and this is now a very streamlined and highly optimized approach. The one request we have is
More informationSTAT3 (py705)/ Pan STAT3 (Human/Mouse/Rat) ELISA Kit
STAT3 (py705)/ Pan STAT3 (Human/Mouse/Rat) ELISA Kit Catalog Number KA2176 96 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Principle of the Assay...
More informationMammalian Cell PE LB
257PR G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Mammalian Cell PE LB Mammalian Cell Protein Extraction & Lysis Buffer (Cat. # 786 180)
More informationItem Catalog Number Manufacturer 1,4-Dithioerythritol (1 g) D9680 Sigma-Aldrich
SOP: Nuclei isolation from fresh mouse tissues and DNaseI treatment Date modified: 01/12/2011 Modified by: E. Giste/ T. Canfield (UW) The following protocol describes the isolation of nuclei and subsequent
More informationFor Research Use Only Ver
INSTRUCTION MANUAL Quick-RNA Miniprep Kit Catalog Nos. R1054 & R1055 Highlights High-quality total RNA (including small RNAs) from a wide range of samples. You can opt to isolate small and large RNAs in
More information[ CARE AND USE MANUAL ] GlycoWorks Single Use Sample Preparation Kit CONTENTS
GlycoWorks Single Use Sample Preparation Kit CONTENTS I. INTRODUCTION General Guideline of Sample Preparation from Glycoprotein to enrich FLR Labeled Glycans Using Reductive Amination Reaction. II. STORAGE
More informationab Lipid Peroxidation (MDA) Assay kit (Colorimetric/ Fluorometric)
Version 10b Last updated 19 December 2018 ab118970 Lipid Peroxidation (MDA) Assay kit (Colorimetric/ Fluorometric) For the measurement of Lipid Peroxidation in plasma, cell culture and tissue extracts.
More informationFor Research Use Only Ver
INSTRUCTION MANUAL Quick-RNA Miniprep Kit Catalog Nos. R1054 & R1055 Highlights High-quality total RNA (including small RNAs) from a wide range of samples. You can opt to isolate small and large RNAs in
More informationDetermination of 6-Chloropicolinic Acid (6-CPA) in Crops by Liquid Chromatography with Tandem Mass Spectrometry Detection. EPL-BAS Method No.
Page 1 of 10 Determination of 6-Chloropicolinic Acid (6-CPA) in Crops by Liquid Chromatography with Tandem Mass Spectrometry Detection EPL-BAS Method No. 205G881B Method Summary: Residues of 6-CPA are
More informationPREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS
TMM,5-2011 PREPARATION OF IF- ENRICHED CYTOSKELETAL PROTEINS Ice-cold means cooled in ice water. In order to prevent proteolysis, make sure to perform all steps on ice. Pre-cool glass homogenizers, buffers
More informationProteomics Grade. Protocol. Catalog # Agilent Technologies. Research Use Only. Not for use in Diagnostic Procedures. Version A, January 2010
Proteomics Grade Trypsin Catalog #204310 Protocol Version A, January 2010 Research Use Only. Not for use in Diagnostic Procedures. Agilent Technologies Notices Agilent Technologies, Inc. 2010 No part of
More informationWestern Immunoblotting Preparation of Samples:
Western Immunoblotting Preparation of Samples: Total Protein Extraction from Culture Cells: Take off the medium Wash culture with 1 x PBS 1 ml hot Cell-lysis Solution into T75 flask Scrap out the cells
More informationThe distribution of log 2 ratio (H/L) for quantified peptides. cleavage sites in each bin of log 2 ratio of quantified. peptides
Journal: Nature Methods Article Title: Corresponding Author: Protein digestion priority is independent of their abundances Mingliang Ye and Hanfa Zou Supplementary Figure 1 Supplementary Figure 2 The distribution
More informationProduct Contents. 1 Specifications 1 Product Description. 2 Buffer Preparation... 3 Protocol. 3 Ordering Information 4
INSTRUCTION MANUAL Quick-RNA Midiprep Kit Catalog No. R1056 Highlights 10 minute method for isolating RNA (up to 1 mg) from a wide range of cell types and tissue samples. Clean-Spin column technology allows
More informationAgilent Protein In-Gel Tryptic Digestion Kit
Agilent 5188-2749 Protein In-Gel Tryptic Digestion Kit Agilent Protein In-Gel Tryptic Digestion Kit Instructions Kit Contents The Protein In-Gel Tryptic Digestion Kit includes sufficient reagents for approximately
More informationASSAY OF SPHINGOMYELINASE ACTIVITY
ASSAY OF SPHINGOMYELINASE ACTIVITY Protocol for Protein Extraction Stock Solution 1. Leupeptin/hydrochloride (FW 463.0,
More informationPhosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining py-mip and TiO 2 affinity resins
Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining py-mip and TiO 2 affinity resins Loreta Bllaci, Silje B. Torsetnes,, Celina Wierzbicka, Sudhirkumar Shinde, Börje Sellergren,
More informationMouse Hepatic Progenitor Organoid Culture: Supplementary Protocols
TECHNICAL BULLETIN Mouse Hepatic Progenitor Organoid Culture: The following are supplementary protocols for the culture of hepatic organoids with HepatiCult Organoid Growth Medium (Mouse) (Catalog #06030).
More informationDetergentOUT Detergent Removal Systems
252PR-04 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DetergentOUT Detergent Removal Systems For the Removal of Detergents from Peptide
More informationProtocol. G-LISA Rac1 Activation Assay Biochem Kit : 24 Assays (Absorbance Based) Cat. # BK128-S. cytoskeleton.com. Cytoskeleton, Inc.
The Protein Experts Protocol Cytoskeleton, Inc. V 8.1 G-LISA Rac1 Activation Assay Biochem Kit : 24 Assays (Absorbance Based) Cat. # BK128-S cytoskeleton.com Phone: (303) 322.2254 Fax: (303) 322.2257 Customer
More informationRayBio Human Phospho-DDR2 (Tyr740) and Total DDR2 ELISA Kit
RayBio Human Phospho-DDR2 (Tyr740) and Total DDR2 ELISA Kit Catalog #: PEL-DDR2-Y740-T User Manual Last revised March 22, 2018 Caution: Extraordinarily useful information enclosed ISO 13485 Certified 3607
More informationFor Research Use Only Ver
INSTRUCTION MANUAL Quick-DNA/RNA Viral MagBead Catalog Nos. R2140 & R2141 Highlights High-throughput, magnetic-bead based purification of viral DNA and RNA from plasma, serum, urine, cell culture media,
More informationNote: During 30 minute incubation; proceed thru appropriate sections below (e.g. sections II, III and V).
LEGEND MAX β Amyloid x 40 LEGEND MAX β Amyloid x 40 ELISA Kit Components and Protocol Kit Components Capture Antibody Coated Plate 1 stripwell plate 1 40 Standard (2) 20μg vial 5X Wash Buffer 125mL Standard
More informationMitochondrial DNA Isolation Kit
Mitochondrial DNA Isolation Kit Catalog Number KA0895 50 assays Version: 01 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials
More informationRayBio Human Phosphotyrosine BTK ELISA Kit
RayBio Human Phosphotyrosine BTK ELISA Kit Catalog #: PEL-BTK-Y User Manual Last revised August 10, 2016 Caution: Extraordinarily useful information enclosed ISO 13485 Certified 3607 Parkway Lane, Suite
More informationSTAT3 (py705) (Human/Mouse/Rat) ELISA Kit
STAT3 (py705) (Human/Mouse/Rat) ELISA Kit Catalog Number KA2175 96 assays Version: 01 Intended for research use only www.abnova.com I. INTRODUCTION STAT3 (py705) (Human/Mouse/Rat) ELISA (Enzyme-Linked
More informationRayBio Human Phospho-DDR1 (Tyr792) ELISA Kit
RayBio Human Phospho-DDR1 (Tyr792) ELISA Kit Catalog #: PEL-DDR1-Y792 User Manual Last revised March 22, 2018 Caution: Extraordinarily useful information enclosed ISO 13485 Certified 3607 Parkway Lane,
More informationDetergentOUT Tween. DetergentOUT GBS10. OrgoSol DetergentOUT
252PR 01 G-Biosciences, St Louis, MO. USA 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DetergentOUT Detergent Removal Systems For the Removal of Detergents
More informationRayBio Human Phospho-DDR1 (Tyr792) and Total DDR1 ELISA Kit
RayBio Human Phospho-DDR1 (Tyr792) and Total DDR1 ELISA Kit Catalog #: PEL-DDR1-Y792-T User Manual Last revised March 22, 2018 Caution: Extraordinarily useful information enclosed ISO 13485 Certified 3607
More informationProtocol. G-LISA RhoA Activation Assay Biochem Kit : 24 Assays (Absorbance Based) Cat. # BK124-S. cytoskeleton.com. Cytoskeleton, Inc.
The Protein Experts Protocol Cytoskeleton, Inc. V. 13.0 G-LISA RhoA Activation Assay Biochem Kit : 24 Assays (Absorbance Based) Cat. # BK124-S cytoskeleton.com Phone: (303) 322.2254 Fax: (303) 322.2257
More informationLys-C/Arg-C, a More Specific and Efficient Digestion Approach for
Lys-C/Arg-C, a More Specific and Efficient Digestion Approach for Proteomics Studies Zhen Wu, Jichang Huang, Jingnan Huang, Qingqing Li, Xumin Zhang *, State Key Laboratory of Genetic Engineering, Department
More informationProduct Use HPSC-CC are for research use only. It is not approved for human or animal use, or for application in in vitro diagnostic procedures.
HPSC-derived Cardiomyocyte Cells (HPSC-CC) Catalog #6240 Cell Specification Human primary cardiomyocytes and cardiac tissue are superior modeling systems for heart disease studies, drug discovery and toxicity
More informationDetermination of β2-agonists in Pork Using Agilent SampliQ SCX Solid-Phase Extraction Cartridges and Liquid Chromatography-Tandem Mass Spectrometry
Determination of β2-agonists in Pork Using Agilent SampliQ SCX Solid-Phase Extraction Cartridges and Liquid Chromatography-Tandem Mass Spectrometry Application Note Food Safety Authors Chenhao Zhai Agilent
More informationProtocol. G-LISA Cdc42 Activation Assay Biochem Kit : 24 Assays (Absorbance Based) Cat. # BK127-S. cytoskeleton.com. Cytoskeleton, Inc.
The Protein Experts Protocol Cytoskeleton, Inc. V 9.5 G-LISA Cdc42 Activation Assay Biochem Kit : 24 Assays (Absorbance Based) Cat. # BK127-S cytoskeleton.com Phone: (303) 322.2254 Fax: (303) 322.2257
More informationDetergentOUT GBS10 Spin Plates
G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name DetergentOUT GBS10 Spin Plates 96-Well Plates for the Removal of Detergents from Peptide
More informationRayBio Human, Mouse and Rat Phospho-NF-kB P65 (Ser536) and Total NF-kB P65 ELISA Kit
RayBio Human, Mouse and Rat Phospho-NF-kB P65 (Ser536) and Total NF-kB P65 ELISA Kit Catalog #: PEL-NFKBP65-S536-T User Manual Last revised October 10, 2017 Caution: Extraordinarily useful information
More informationab Human Citrate Synthase (CS) Activity Assay Kit
ab119692 Human Citrate Synthase (CS) Activity Assay Kit Instructions for Use For the measurement of mitochondrial citrate synthase (CS) activity in Human samples This product is for research use only and
More informationby nexttec TM 1 -Step
Protocol DNA Isolation from Bacteria by nexttec TM 1 -Step - nexttec cleancolumns - Cat. No. 20N.010 Cat. No. 20N.050 Cat. No. 20N.250 Version 1.0 For research only Principle nexttec 1 -Step is the easiest
More informationPRODUCT INFORMATION & MANUAL
PRODUCT INFORMATION & MANUAL 0.4 micron for Overall Exosome Isolation (Cell Media) NBP2-49826 For research use only. Not for diagnostic or therapeutic procedures. www.novusbio.com - P: 303.730.1950 - P:
More informationMagCapture Exosome Isolation Kit PS Q&A
MagCapture Exosome Isolation Kit PS Q&A Specifications and performance P.1 Comparison of the conventional method P.2 Operation methods and composition P.4 Amount of starting sample P.5 Analysis after exosomes
More informationMammalian Membrane Protein Extraction Kit
Mammalian Membrane Protein Extraction Kit Catalog number: AR0155 Boster s Mammalian Membrane Protein Extraction Kit is a simple, rapid and reproducible method to prepare cellular protein fractions highly
More informationWhole Mount Drosophila Embryo In Situ Hybridization with RNA probes 2/5/2001 Leslie Vosshall
Whole Mount Drosophila Embryo In Situ Hybridization with RNA probes 2/5/2001 Leslie Vosshall DAY ONE All incubations are done at room temperature unless otherwise noted. All solutions and all containers
More informationSTAT1 (ps727) (Human/Mouse) ELISA Kit
STAT1 (ps727) (Human/Mouse) ELISA Kit Catalog Number KA2171 96 assays Version: 01 Intended for research use only www.abnova.com I. INTRODUCTION STAT1 (ps727) (Human/Mouse) ELISA (Enzyme-Linked Immunosorbent
More informationHuman TSH ELISA Kit. User Manual
Human TSH ELISA Kit User Manual Catalog number: GTX15585 GeneTex Table of Contents A. Product Description... 2 B. Kit Components... 3 C. Additional Required Materials (not included)... 3 D. Reagent Preparation...
More informationProtease Assay. (Cat. # ) think proteins! think G-Biosciences
389PR 01 G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name Protease Assay (Cat. # 786 028) think proteins! think G-Biosciences www.gbiosciences.com
More informationData Sheet TIGIT / NFAT Reporter - Jurkat Cell Line Catalog #60538
Data Sheet TIGIT / NFAT Reporter - Jurkat Cell Line Catalog #60538 Background: TIGIT is a co-inhibitory receptor that is highly expressed in Natural Killer (NK) cells, activated CD4+, CD8+ and regulatory
More informationProtein Dephosphorylation Methods
Protein Dephosphorylation Methods Phosphospecific antibodies are designed to differentiate between the phosphorylated and the non-phosphorylated states of a protein. The method to determine if or how well
More informationStriatal Neuron Medium Kit
Striatal Neuron Medium Kit Product Information What are included in the Striatal Neuron Medium Kit (ax0333): 2x 250 ml Striatal Neuron Basal Medium (Store at 4 o C upon receipt) 2x 7.5 ml Striatal Neuron
More informationMitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit
PROTOCOL Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit DESCRIPTION Mitochondrial Trifunctional Protein (TFP) Protein Quantity Microplate Assay Kit Sufficient materials
More informationXCF TM COMPLETE Exosome and cfdna Isolation Kit (for Serum & Plasma)
XCF TM COMPLETE Exosome and cfdna Isolation Kit (for Serum & Plasma) Cat# XCF100A-1 User Manual Store kit components at +4ºC and +25ºC Version 1 2/2/2017 A limited-use label license covers this product.
More informationab Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric)
ab156064 Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric) Instructions for Use For the quantitative measurement of Histone Deacetylase activity in cell lysates This product is for research
More informationProduct Contents... 1 Specifications... 1 Product Description... 2 Buffer Preparation... 3 Protocol Appendix... 5 Ordering Information...
INSTRUCTION MANUAL Quick-DNA/RNA Pathogen MagBead Catalog Nos. R2145 & R2146 Highlights High-throughput, magnetic-bead based purification of pathogen (virus, bacteria, protozoa) DNA/RNA from a wide variety
More informationCaution: For Laboratory Use. A product for research purposes only. Eu-W1024 ITC Chelate & Europium Standard. Product Number: AD0013
TECHNICAL DATA SHEET Lance Caution: For Laboratory Use. A product for research purposes only. Eu-W1024 ITC Chelate & Europium Standard Product Number: AD0013 INTRODUCTION: Fluorescent isothiocyanato-activated
More informationHuman ipsc-derived Ventricular Cardiomyocytes. Protocol version 3.1
Human ipsc-derived Ventricular Cardiomyocytes Protocol version 3.1 Protocol version 3.1 Table of Contents Product Information 2 Recommendations 2 Preparing Cardiomyocyte Maintenance Medium 3 Cardiomyocyte
More informationCharacterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry. Supporting Information
Characterization of Disulfide Linkages in Proteins by 193 nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry M. Montana Quick, Christopher M. Crittenden, Jake A. Rosenberg, and Jennifer S. Brodbelt
More informationGlobal Histone H3 Acetylation Assay Kit
Global Histone H3 Acetylation Assay Kit Catalog Number KA0633 96 assays Version: 06 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 Principle
More informationPRODUCT: RNAzol BD for Blood May 2014 Catalog No: RB 192 Storage: Store at room temperature
PRODUCT: RNAzol BD for Blood May 2014 Catalog No: RB 192 Storage: Store at room temperature PRODUCT DESCRIPTION. RNAzol BD is a reagent for isolation of total RNA from whole blood, plasma or serum of human
More informationSupplementary material: Materials and suppliers
Supplementary material: Materials and suppliers Electrophoresis consumables including tris-glycine, acrylamide, SDS buffer and Coomassie Brilliant Blue G-2 dye (CBB) were purchased from Ameresco (Solon,
More informationDetailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit
be INSPIRED drive DISCOVERY stay GENUINE APPLICATION NOTE Detailed Characterization of Antibody Glycan Structure using the N-Glycan Sequencing Kit Beth McLeod, New England Biolabs, Inc. Materials Remicade
More informationFor Research Use Only Ver
INSTRUCTION MANUAL Quick-DNA/RNA Pathogen Miniprep Catalog Nos. R1042 & R1043 Highlights Spin-column purification of pathogen (virus, bacteria, protozoa) DNA/RNA from a wide variety of vectors (mosquitoes,
More informationThe Immunoassay Guide to Successful Mass Spectrometry. Orr Sharpe Robinson Lab SUMS User Meeting October 29, 2013
The Immunoassay Guide to Successful Mass Spectrometry Orr Sharpe Robinson Lab SUMS User Meeting October 29, 2013 What is it? Hey! Look at that! Something is reacting in here! I just wish I knew what it
More information