Hey, i've been doing a western blot like from 3 weeks ago, and i can't get good results, i'm getting this type of runs, and i don't know what can it be, maybe degraded proteins?.
I'll be very grateful if someone can help. Thanks
Hi Marcelo Díaz
From where did you isolate your proteins? Have you blotted them? If so, can you the final images of the protein bands?
i isolated from bacteria pellet ,when blotted (western) it showed no signal. I also did a dot blot and it showed signal
There are a few things to troubleshoot. But the first things that come to mind are:
1. Can you see protein transferring to the membrane? To check this, stain the membrane with the removable stain called ponceau S and stain the gel with coomassie. You should see no bands on the gel but red bands on the membrane.
2. looks like your protein either overloaded or degraded on the gel. Do you include protease inhibitor in your protein prep procedure? Try loading less and see if you can resolve some protein bands. if you still see a smear, then I think you protein degraded (either via oxidation or protease). Next time include protease inhibitor and/or DTT or BME reducing agents in your protein prep.
hey, i've added 5 uL of protease inhibitor cocktail, which includes
AEBSF hydrochloride: serine protease inhibitor (0.5 mM) Aprotinin (bovine lung): serine protease inhibitor (0.00015 mM) E-64: cysteine protease inhibitor (0.001 mM) EDTA disodium salt: metalloprotease inhibitor (0.5 mM) Leupeptin hemisulfate: serine/cysteine protease inhibitor (0.001 mM)
maybe too much?
I also did a ponceau Stain and only got 1 band in the middle top of the membrane
Marcelo Díaz the most possible problem is either your proteins are degraded or you have too low concentration of protein in your samples.
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