UV Tracer TM Maleimide NHS ester

Transcription

1 UV Tracer TM Maleimide HS ester Product o.: 1020 Product ame: UV-Tracer TM Maleimide-HS ester Chemical Structure: Chemical Composition: C 41 H Molecular Weight: Appearance: Storage: Yellow oil Upon receipt store at -20 C. Product shipped at ambient temperature Description UV Tracer TM Maleimide HS ester is a UV Vis traceable, heterobifunctional crosslinker with hydroxysuccinimide (HS) ester and maleimide groups that allow covalent conjugation of amine and sulfhydryl containing molecules. Crosslinkers having UV Vis traceable spacers are convenient alternatives to reagents with purely hydrocarbon, or PEG spacer arms. The efficiency of maleimide incorporation can easily be measured in minutes by means of non destructive UV Vis spectroscopy providing greater precision in optimization and characterization of crosslinking applications. hydroxysuccinimide (HS) esters react with primary amines at ph 7 9 to form amide bonds, while maleimides react with sulfhydryl groups at ph to form stable thioether bonds (Figure 1). In aqueous solutions, hydrolysis of the HS ester is a competing reaction whose rate increases with ph. The maleimide group is more stable than the HS ester group but will slowly hydrolyze and also lose its reactivity toward sulfhydryls at ph values greater than 7.5. For these reasons, conjugation experiments involving this type of heterobifunctional crosslinker are usually performed between ph

2 Protein-H 2 H 2 Step 1 Activation of Protein-H 2 with UV-Tracer TM Maleimide-HS ester H Maleimide-activated protein 1 HS Step 2 Conjugation of Maleimide-activated Protein-H 2 with Protein-SH H Crosslinked proteins S Directly measure activation level of Protein-H 2 by non-destructive UV-Vis scan Figure 1. Schematic representation of crosslinking by reaction of UV Tracer TM Maleimide HS with amine and sulfhydryl molecules Important Product Information UV Tracer TM Maleimide HS ester reagent is a viscous pale yellow liquid that is difficult to weigh and dispense. To facilitate handling, make a stock solution immediately before first use by dissolving the crosslinker in anhydrous water miscible solvent, such as DMS. Minimize reagent exposure to moisture because the HS ester reactive group is susceptible to hydrolysis. With proper handling under moisturefree conditions the unused stock solution is stable for one month at 20 C. Avoid buffers containing primary amines (e.g., Tris or glycine) and sulfhydryls during conjugation because they will compete with the intended reaction. If necessary, dialyze or desalt samples into an appropriate buffer such as phosphate buffered saline (PBS). Molecules to be reacted with the maleimide moiety must have free (reduced) sulfhydryls. If absent, sulfhydryls can be added to biomolecules using succinimidyl S acetylthioacetate (SATA). Be aware that some proteins can be inactivated by reduction of their disulfide bonds. Adding EDTA to 1 5mM helps to chelate divalent metals, thereby reducing disulfide formation in the sulfhydryl containing protein. For maximum reproducibility, it is preferable to buffer exchange proteins into modification buffer. This simple procedure guarantees maximum consistency during the labeling reaction. Procedure for Two-step Protein Crosslinking Generally, a 10 to 50 fold molar excess of UV Tracer Maleimide HS crosslinker over the amount of amine containing protein results in sufficient maleimide activation to enable efficient conjugation to sulfhydryl containing proteins. Highly dilute protein solutions require a high molar excess of reagent to achieve adequate activation. Empirical testing is necessary to determine activation levels and final conjugation ratios that are optimal for the intended application.

3 Additional Materials Required Water miscible organic solvent such as dimethyl sulfoxide (DMS) or dimethyl formamide (DMF) Conjugation buffer: Phosphate buffered saline ph 6.5 or 20 mm MPS, 100 mm acl ph 6.5. Modification buffer: Phosphate buffered saline (PBS) at ph 7.5. Quenching buffer: 1 M Tris HCL, ph 8.0 (optional) Desalting Columns (spin or gravity flow) TCEP HCL and 500 mm EDTA (ph 8.0) Protein 1 (Protein-SH) Reduction (1-3 mg/ml) 1. Prior to conjugation, proteins containing disulfide bonds or free cysteines must be reduced with TCEP (or DTT) to insure proper labeling. 2. Prepare a stock solution of TCEP HCL (105 mm) in ultrapure water, vortex to dissolve crystals completely. 3. Add a volume of 500 mm EDTA to protein 1 solution (1 3 mg/ml) to obtain a 1 mm final concentration, pipette the mixture up and down several times to mix. 4. Add a volume of TCEP solution (105 mm) to protein 1 solution (1 3 mg/ml in Conjugation buffer) to obtain a final concentration of 5 mm TCEP, pipette up and down several times to mix. 5. Allow the reaction to incubate for 30 minutes. 6. Buffer exchange TCEP reduced protein 1 into Conjugation buffer containing 1 mm EDTA using an appropriately equilibrated desalting column, pipette up and down several times to mix. Protein 2 (Protein-H2) Modification (2-10 mg/ml) 1. Prepare protein 2 in Modification buffer at µm (e.g mg in 1 ml for a 50 kda protein). ote protein 2 (protein used in excess) is generally prepared at higher concentrations than protein 1 (Protein SH). 2. Immediately before use prepare a 100 mm stock solution of UV Tracer TM Maleimide HS ester by dissolving reagent in anhydrous DMS. Vortex for 1 2 minutes to mix well. 3. Add a volume of UV Tracer TM Maleimide HS ester to Protein 1 to obtain a fold excess. ote If [protein] < 1mg/mL use X, 1 4 mg/ml use 20X, 5 10 mg/ml use 10X. 4. Incubate the reaction at room temperature for 60 minutes. 5. Remove excess un reacted reagent with an appropriate desalting column and immediately proceed to determine the concentration of Protein 1 and Protein 2.

4 Determine Concentration Protein 1 and Protein 2 (mg/ml) 1. Set a spectrophotometer to simultaneously measure the absorbance of Protein 1 and Protein 2 at 280 nm and 350 nm. ote for concentrated protein solutions first dilute an aliquot of each protein into Modification buffer (e.g fold, 0.1 to 1.0 AU). When using a anodrop TM spectrophotometer (or similar) dilutions are not required. 2. Calculate Protein 1 and Protein 2 concentrations (see calculations, Eq. 1 and Eq.2). 3. Immediately proceed to protein protein conjugation. ptional: The degree of maleimide activation on protein 2 can be quantified prior to conjugation. See calculations. Protein-Protein Conjugation 1. Add a volume of reduced Protein 1 (limiting protein) to Protein 2 (excess maleimide protein) in a molar ratio corresponding to that desired for the final conjugate (e.g. 1:3). See calculations, Eq. 6, example2) 2. Incubate the reaction mixture at room temperature for 2 12 hours or overnight at 4 C. 3. Purify conjugate away from excess un reacted Protein 2 using an appropriate purification method (e.g. size exclusion, ion exchange, mixed mode, etc.). For less demanding applications, purification of protein protein conjugates may not be required. 4. Conjugate is now ready for use. Calculations Calculate protein 1 concentration with Equation 1:. 1 / 1 Calculate protein 2 concentration with Equation 2:. 2 / 2 1% % 10 ptional Calculate degree of maleimide labeling on protein 2 with Equations 3, 4, and 5:

5 ,500 M 1 cm % M 1 cm 1 10 E1% = A 280 of a 10 mg/ml solution M.W. = protein molecular weight Example 1: Determine Concentration of Protein 1, Protein 2 and DL Protein 1 (GIgG 0.5 ml) 2.5 mg/ml was reduced in 5 mm TCEP for 30 minutes, subsequently desalted into 20 mm MPS, 100 mm acl, ph 6.5. At the same time, Protein 2 (BSA mg/ml) was labeled using a 10 fold molar excess UV Tracer Maleimide HS ester reagent for 1 hr, then desalted. The A 280 of protein 1 (1:10 dilution) was determined to be The A 280 and A 350 (1:10 dilution) of Protein 2 was and , respectively. E1% BSA = 6.7 (i.e. 44,500 M cm ) E1% GIgG = 13.6 (i.e.204,000 M 1 cm 1 ) Calculate protein 1 concentration after reduction (mg/ml) 1 / / Calculate protein 2 concentration after maleimide labeling (mg/ml) 2 / / Calculate maleimide degree of Llbeling (# maleimide/bsa) as follows: , ,

6 Example 2: Determine volume of Protein 1 (limiting protein) to Mix with Protein 2 (excess protein). Calculate Volume of Protein In Excess (V excess ) to Mix with Volume of Limiting Protein (V limiting ) During a Conjugation by Equation 6: 6 Calculate volume of excess protein 2 (BSA 2.64 mg/ml, 66.4kDa) to mix with 0.1 ml reduced protein mg/ml, 150 kda) using a 3 fold excess: / / 66, , Therefore, 0.10 ml of BSA 2.64 mg/ml must be added to 0.1 ml reduced IgG (2.0 mg/ml) to initiate protein protein conjugation reaction at 1:3 stoichiometry. UV-Vis Spectra of UV-Tracer Maleimide HS Labeled BSA Three bovine serum albumin samples were prepared in 100 mm sodium phosphate, 150 mm sodium chloride, ph 7.5 at 2.5 mg/ml. Samples were subsequently labeled using a 5 fold, 10 fold, or 20 fold excess UV Tracer TM Maleimide HS ester for 60 minutes at room temperature. After removing excess reagent each sample was scanned on a spectrophotometer ( nm). Resulting A 280 and A 350 values were used to quantify degree of maleimide labeling X Absorbance X 5X 20X (DL 9.0) 10X (DL 5.5) 5X (DL 3.4) Wavelength (nm) Figure 2. Superimposed UV scans of BSA labeled with UV Tracer TM Maleimide HS Ester at 5, 10, and 20 fold mole equivalent excess reagent. The corresponding degrees of labeling (DL) were 3.4, 5.9, and 9.0, respectively.

7 Example Protein-Protein Conjugation Duplicate BSA (2.5 mg/ml) samples were labeled using 20X UV Tracer TM Maleimide HS Ester, the resulting maleimide BSAs (DL 9.0) were conjugated (in excess) to TCEP reduced Goat IgG (1:4 ratio). Aliquots from the duplicate crude reactions were removed after 3 hours and analyzed by SDS PAGE (Figure3) kda 250 kda 180 kda 130 kda 100 kda 70 kda 50 kda 40 kda IgG BSA conjugates IgG SH Excess BSA Maleimide (DL 9.0) 1. High M.W. protein marker 2. Goat IgG (non reduced) + BSA (unmodified) Unlabeled Controls 5 µg 3. Goat IgG (TCEP reduced) + BSA UV Tracer TM Maleimide (DL 9.0) 1:4 Ratio 5 µg 4. Goat IgG (TCEP reduced) + BSA UV Tracer TM Maleimide (DL 9.0) 1:4 Ratio 5 µg Figure 3. SDS PAGE (non reducing) analysis of IgG BSA protein protein conjugates