Protein SDS-PAGE thick bands / smear
Posted 22 October 2010 - 04:24 PM
Attached is an example of a cropped gel. The line going from left to bottom right is an artifact of scanning the gel.
lanes with smeared protein are what I'm working on, crisp lanes are standards.
10% gel, Coomassie stained.
In an alternate problem, there is the outline of the lane that is a light smear. I've read that decreasing salt might fix that; corroboration? and this Sample buffer doesn't have NaCl added to it, what should I decrease?
Sample buffer formula:
0.004% Bromophenol Blue
Posted 23 October 2010 - 04:08 AM
Edited by donny, 23 October 2010 - 04:13 AM.
Posted 25 October 2010 - 07:17 AM
that pH difference is a concern, it should be 6.8 for the laemmli buffer system (at least for the stacking gel buffer).
more protein appears to be loaded than necessary. how much did you load?
salt is contributed by the buffer that the sample is in before adding the loading buffer.
addition of urea can sharpen the bands. add it to the gel formula, not just the sample buffer.
you can also sharpen the bands by using a gradient gel.
genius does what it must
i do what i get paid to do
Posted 26 October 2010 - 09:16 AM
stacking gel recipe:
I thought the gel or running buffer wouldn't be the issue because the standard looks just fine.
I'll try making new sample buffer at pH 6.8
no chance of DNA contamination - these are purchased pure proteins.
currently loading 4µg per well
I ran a range of proteins (0.8-4µg/well), and lower protein amounts with pH 6.8 do give smaller band (but pH 7.0 doesn't). I think that's what I was missing the last time I tried decreasing the amount. Looks like I might need to decrease the amount of protein in each well even more. I will try a 25-400ng/well range.
Posted 27 October 2010 - 09:18 AM
lane 1 = 40 ng
lane 2 = 80 ng
lane 15 = 500 ng
lane 5-10 is exactly what I'm looking for (200-400ng/well)
thanks for the help!