partial proteolysis by trypsin - (Jun/27/2006 )
does anybdy do a partial proteolysis by trypsin. If
yes, can you plz send me the exact protocol word by
word.If the fragments are very small (less than 2kd),
how do they resolve on SDS-PAGE or tricine-PAGE.
i have done this before and it depends on the amount of sites in your protein
do you know the exact amount of protein that you have??
its best to do a time course with a small amount of sample (in 200 microl and add 20 microl trypsin, proteomics grade 100ng/ml) i incubate on the bench for 1, 2, 3, 4, 5, 6 and 16 hours. Take aliquots and add trypsin inhibitor as heating doesnt inactivate it completely nor does freezing.
analyse by sds-page for cleavage and select appropriate time point and scale up for remaining sample
2kD fragments dont resolve well by sds-page in my experience i would use reversed phase hplc instead, the fractions can then be dried and sent for sequencing.
hope this helps
That all sounds good; you can resolve 2 kDa on Tris-Tricine gels.