Background: NTCB cleaves the peptide bond at the N-terminus of Cys by cyanilation of thiol at higher pH.This is commonly used for analytical purposes (massspec analysis etc.) I have been trying to cleave my super hydrophobic aggregated protein with NTCB following the standard protocol (protein is reduced with 1mM DTT,5 mM NTCB is added,cyanilation reaction carried out at pH 8.0 at 40 0C,later shifted to pH 9.0 for peptide bond cleavage at 50 0C,buffer exchange done with centricons)
Hurdle: I don’t see any cleavage when the sample after cleavage is run on an 18% tris Tricine gel. I have tried longer incubation times (overnight) .The possible solutions that I am working on at the moment is addition of Urea (my protein stays active at 1M),removal of DTT( as it has 2 thiol groups and NTCB can act on it,and I dont think my protein forms disulphide bonds ) .
Does the modification at the C-terminal make a difference in the gel migration pattern?My goal is to achieve close to 100% cleavage efficiency. Any suggestions will be appreciated..Thanx!
Do you know whether the cysteine is reactive in the native protein? In general, most cysteines are buried, and the cleavage reaction proceeds only after denaturation with SDS, urea, or guanidine. The pH and temperature that you are using should be OK.
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