Method Notes:

Step 1   Some proteins may require a denaturant to expose buried disulfides.  If this is the case Urea (1-8M) or Guanidine HCl (1-6M) may be used in this RCM buffer.  If urea is used, avoid heating the reduction reaction because this can promote carbamylation.

Step 2 Some resistant proteins benefit from; higher concentrations of the reducing agent, addition of a chaotrope and increased temperature.  During the reduction of a very resistant protein we have used up to 80 mM dithiothreitol , 6 M guanidne HCl, at 60°C for 60 min..  

Step 4  Make the iodoacetic acid up in 1M NaOH.  The iodoacetic acid, dry reagent, should be a white flaky crystalline material.  It should not be brown but it can be slightly yellow.  The color in the reagent is free iodine which can oxidize your protein or peptide.  If the reagent is slightly yellow the excess DTT in the reduction will quench this effect.  Always make and use the iodoacetic acid reagent fresh.  Be careful with this reagent it's dangerous, don't alkylate yourself !   
This is how we weigh out our IAA:  
(A)  Tare the receiving container (1.5 ml capped tube) on the balance.  
(B) Add a small chip of  iodoacetic acid in a fume hood to the tared tube.
(C) Weigh the capped tube on the balance. 
(D) Add the appropriate amount of 1 N NaOH to the tube in the fume hood. 
This may be overly cautious, but better safe than sorry.  Always wear gloves, safety glasses and a lab coat.  Do not let the carboxymethylation go for an extended period of time, it is possible to over alkylate.  There are reports of histidine, methionine and tryptophan being alkylated admittedly, at lower efficiencies.  The cysteine reaction is almost instantaneous, therefore 30 min in the dark, should be way more than enough time.  Again, if you have the time, method development is in order.

Steps 5 and 6  Steps 5 and 6 can be bypassed with a quick size exclusion or reverse phase purification. The down side of dialysis is that it can take as long as 24hrs.  The advantage of size exclusion or dialysis is that the protein can be exchanged in to the enzyme digest buffer.

Deamidation Risk  The RCM reaction is carried out at high pH and the reduction is often carried out at elevated temperature and pH which makes deamidation of proteins during the procedure likely.  To reduce the likelihood of deamidation keep the procedure as short as possible and avoid or reduce the heating time on the reduction.  If artifctual deamidation is a real concern for you the pH of the RCM procedure can be reduced but some method development and validation will need to be done on your part. Most likely the method you develop will be specific to your particular protein. 

Dithiothreitol

S-Carboxymethylated 
Cysteine

.

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Last updated:  Tuesday, January 19, 2016 02:49:30 PM