I am expressing a part of a protein with a fusion tag. After cleaving the fusion tag by digestion with protease, the target protein has weight of about 2 kD. Can it be detected by SDS-PAGE? If not, what should I do confirm the presence of target protein after digestion?
2 kDa is hardly a protein, at around 20 aa it should be more properly called a peptide.
Although I agree with Adam Shapiro that it can be detect with SDS-PAGE, I would rather go with MALDI as suggested by Ravi Kishore: desalt your sample with a C18 zip-tip and spot directly on a MALDI plate, much faster and more accurate Mw determination.
Yes, it can be detected. The best type of gel to use is a Tris-Tricine gel:
https://www.ncbi.nlm.nih.gov/pubmed/2449095
2 kDa is hardly a protein, at around 20 aa it should be more properly called a peptide.
Although I agree with Adam Shapiro that it can be detect with SDS-PAGE, I would rather go with MALDI as suggested by Ravi Kishore: desalt your sample with a C18 zip-tip and spot directly on a MALDI plate, much faster and more accurate Mw determination.
It is possible to detect peptides but there can be problems when using standard staining procedures involving fixing and staining with dyes that are noncovalently bound to the peptides. Peptides may not be fixed well and can be washed out of the gel during the staining procedure. The smaller and more soluble the peptides the more likely it is that peptide will be lost and either not detected or detected with low sensitivity. One way to overcome this problem is to label the peptide with a small fluorescent molecule before the electrophroesis. The peptide can then be viewed in the gel while still in the glass electrophoresis cassette. Peptides can be labelled on either amino groups ( at N-terminal or on lysine side chain amino) or on cysteine sulphydyl (-SH) group.
Reagents and some methods for labelling SH groups are given in US patents:- US2005/0224354 and US5,320,727
and in:- Jackson, P., & Urwin, V.E. Analytical Biochemistry 209(1):57-62 · March 1993.
They can also be obtained from the numerous suppliers of -SH labelling fluorophores .
Fluorophores and protocols for labelling amino groups can be obtained from the numerous suppliers of these reagents.
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