how much concentration of tcep require to reduce the protein without interfering the maleimide cysteine conjugation reaction?
Here is a protocol for maleimide labeling of proteins following TCEP reduction. It suggests a 100X molar excess of TCEP.
https://www.lumiprobe.com/protocols/protein-maleimide-labeling
Try using an excess (100x)! Hope your experiment works! Good luck!!
P.S.- check out if this procedure in this paper, it might help since TCEP is used in a labeling buffer (Article Multiple conformational states of DnaA protein regulate its ...
)
I'm sorry Shoyab, this paper may be of more use. Article Site-Specific Fluorescence Double-Labeling of Proteins and A...
Specifically mentions the use of 0.5 and 0.1 mM TCEP usage.
I was trying to label my virus which has 180 cysteine sites available. Prior to labelling, the virus is present in buffer ( tried both pH6 and 7). I treated them with 10x tcep for 1 hr( degassed and inert buffers) and then labelled them with Oregon green 488 maleimide dye(5-20x excess dye). I tried few times , but I am getting very poor labelling. Is it because the tcep concentration was low?
Suggestions please!!
According to my standardized protocol I have used 20X TCEP and incubate for 20 minutes and I did get a very good labeling ratio. But because my protein has only one cysteine. So maybe TCEP concentration of yours is very low. Beside TCEP I want to know your incubation procedure after labeling?
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