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Product Code	CJ30C
Buffer	Protein is supplied in a solution of 50 mM MES, 2 mM EDTA and 0.5 µg/ml pentachlorophenol
Storage:	4°C: DO NOT FREEZE

SUGGESTIONS FOR USE

To conjugate to sulfhydryl groups in your protein, reduce the sulfhydryl by using the protocol shown in our Conjugation Kits located on our webpage at:

 http://www.prozyme.com/pdf/pj25kit.pdf

Cysteines may be introduced into your protein by thiolation with 2-iminothiolane:

Thiolation of Unspecified Protein with 2-iminothiolane
 

1. Your protein should be in PBS or other non-amine containing buffer between pH 7 and pH 9.  For best results, the starting concentration of your protein should ideally be greater than 5 mg/ml so that after the desalting column in step 3 the protein is at a concentration greater the 1 mg/ml.

2. Prepare a 1 mg/ml stock of iminothiolane (MW = 137 Daltons) in PBS and immediately add enough of the stock to give about a 5:1 molar ratio with your protein.  For instance to 1 mg of an antibody (MW = 150,000 kD) add 5 µl of iminothiolane stock per mg of antibody.  Mix and incubate 45 minutes at room temperature.  After the initial mixing, try to avoid further mixing to prevent air oxidation of the thiol groups.

3. Desalt your thiolated protein into exchange buffer (50 mM MES, 2 mM EDTA and 0.5 µg/ml pentachlorophenol as an antimicrobial agent, adjusted to pH 6.0 with NaOH.)

4. Immediately combine your thiolated protein with SMCC-Streptavidin (MW = 52,000 kD).  Start by mixing the two in a 1:1 molar ratio.  Incubate the mixture for 1-2 hours at room temperature.

5. (Optional) Block further reaction by adding 10 µl of 10 mg/ml N-ethyl-maleimide dissolved in Dry DMSO per ml of reaction.  Incubate the mixture for 20 minutes at room temperature.

6. Use a desalting column to exchange the conjugate into the desired storage buffer.  Otherwise, if desired, purify the conjugate by size exclusion chromatography.  If the protein is between 50,000 kD and 250,000 kD, use a matrix like Biogel A0.5 M (Bio-Rad, fractionation 10,000-500,000 for globular proteins).  Pre-equilibrate the column with whatever buffer you want to store the conjugate in (excluding any large MW stabilizers such as BSA).  The conjugate should run as 1 or more peaks which elute before any unconjugated remnants of Streptavidin or your protein.

7. To optimize the yield and size distribution of the conjugate, alter the amount of iminothiolane in step 2 above or the ratio of Streptavidin to your protein used in step 4.

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Copyright © 2007 Rev. 12/05/07