expanded band of the purified protein on silverstain gel (even in low expression - (Dec/04/2008 )
I am trying to purify a his-tag protein and when I do silverstain I find an expanded band on the expected molecular weight. This is not due to high amount of the expression. In this small smear I cannot see any other bands. can anyone have an idea what can this be?
Thanks in advance
Thanks in advance
The band you see could be an artefact of silver staining - what size does this smear come out at?.
When we silver stain our SDS-PAGE gels we see a smear right across the gel (even in empty lanes) at about 55 kDa. I think this is due to high levels of 2-mercaptoethanol (we use 1% in our sample buffer). You could try using less mercaptoethanol or try 1 mM DTT instead and see if you still get this band.
This smear comes out at about 11-17kDa and the 2-mercaptoethanol that we use is 10% in our sample buffer. Thank you for your help and sorry for the delay.
does the band look like it is in the lane or does it just run all the way across?
if it runs all the way across then it could be an artifact from your running buffer. to eliminate this, either run the gel longer (this could cause you to overrun the gel a little) or prepare fresh running buffer using fresh components (especially sds).
could you post a picture of your gel so that we could make a better educated guess?
Yes of course! Thank you for your reply and sorry for the delay! I post a picture of a gel where you can see the smear that appears. I hope I manage to send it. If not I will try again.
a couple of things:
first, the gel does look like it can be run further. what is the gel percentage? your higher standards are bunched at the top. could you run a gradient?
second, what is your sample in (besides the loading buffer)? your standards look like they have been affected by the adjacent lane. excess salt or other factors can cause this.
The percentage of the gel is 17.5%, so I could let it run further and as for the sample, except for the loading buffer it also contains HEPES 20mM, NaCl 250mM and mercaptoethanol 10%. Thanks a lot!