Peptide Isolation from lysates - Need help isolating small peptides from lysate (Dec/06/2006 )

Hello all,

I am attempting to isolate small peptides (4-6 kDa) from rat brain (a few different regions) lysate. I hope to use the isolated peptides for Western blotting.

I have resorted to trying this method because I have not had a great deal of success with blotting using the entire lysate.

Any and all advice is welcome and appreciated. Thanks in advance.

Perhaps you could use dialysis. The small peptides would slip through the net whilst the bigger proteins would be retained. Alternatively, precipitate out the bigger peptides and proteins with PEG or ammonium sulphate. This might be harsher on the peptides you want to analyse, mind you.

Time ago I've read rather old papers on pituary peptides purification by solid phase extraction on reversed phase and ion exchange cartridges. Try to search by name "Bennet" from late 70's to end of 80's, or send me private message and I'll do my best to send you some info.

I would use 10K MWCO centricon, then lyophilize the flow thru to concentrate....if I need specific peptide, reverse phase fractionation.

Thanks all, I appreciate the advice. I'll first try the microcon filters followed by lyophilization because that seems to be the most straight forward and practical for my lab. If that doesn't work, I have a colleague who does dialysis regularly, so I'll enlist his help. Unfortunately, we are mostly a molecular lab so K.B.'s great suggestions are not possible for me now, but I will keep them in the back of my mind if the filters and dialysis do not work out.

Thanks again everyone.