Hydrolysis is not performed by sds-page. Reduction happens if using dithiotheitol or mercaptoethanol. If you are concerned about boiling the sample, do not do it; 15 min at 37C is enough for denaturation. Are you using protease inhibitor during lysis and subsequently?

SDS whach effect by high temperature and pH

Dear Sam

as Omar suggest, i think that is more probable that the degradation happen during the sonication that during the sds page sample preparation.

i runned undreds purified proteins in sds page (also dusiin with gfp, gst, mbp etc) and i never observed rhis phenomena.

loading buffer with sds and boiling generally froze the situation.you can try to avoid the boiling if you want.

In the most of cases the degradation happen beetwen cell lysis and sample preparatiom for sds page when your protein become in contact with the orher e.coli proteins ( and proteases )

did yoy used protease inhibitors or edta in your lysis buffer?

did you pay attention to design a sonication cicle that mantaining the sample in ice allow to not mantain loe the sample temperature?

good luck

Manuele

Sonication can sometimes cause protein loss, I found a french press gentler and more effective.

Did you include protease inhibitors in your lysis buffer?

Incubation in SDS-sample buffer unfolds proteins and thus makes them more susceptible to proteolytic attack, unfortunately some proteases are active even in sample buffer.

Thanks everyone for the response.

A few remarks on my situation:

(1) yes I used a combination of PMSF, benzamidine and inhibitor cocktail in a cold lysis buffer, but still the peptide part was seen lost.

(2) sonication - at first we suspect sonication was an issue. Instead we then used 0.2% Tween-20 and passed through a 25G syringe several times. Still degradation occured.

(3) We sort of confirm that the in the whole cell lysate the GFP-peptide was intact. (a considerable amount of GFP-peptide along with some degradation product viz. GFP were observed after SDS-PAGE with out boiling.)

Assuming that the fusion product is formed and stable in E. coli (and those are big "if"), you can only try to find an inhibitor that protects your protein from degradation. I once had a case where degradation stopped only after addition of aminocaproic acid, an inhibitor of Ser-proteases which in theory should have been blocked already by PMSF.

Protein purification is still more art than science.

For E.coli lysis we use french press. It is more gentle. I agree that you can loose your protein upon sonication. Sometimes optimizing your expression and buffer systems can help. Usage of synthetic genes optimized for expression in E.coli.

Good luck

Jana