Recently I tried a western blot on various human muscle samples. Unfortunately, some of the samples migrated like the fourth sample you see in the picture. It seems like the proteins in the sample are too aggregated, but I really don't know what to do to "unpack" them and no one in my lab can give me any advice about it. I used RIPA lysis buffer to lyse the muscle sections, and broke them down by freezing and thawing them a few times.
Any advice would be immensely appreciated.
Have you tried boiling the samples before running them? I incubate my Western samples at 95 C for 5 mins before running them on a SDS-PAGE gel.
@ Galletta:- Based on my experience with similar issues, some important points that you may consider can be:
(1) Please check the thickness/density of the samples for which you see this problem. If the sample is dense/thick, it will not run properly and will end up generating band with a shape that you have depicted in your blot.
(2) Make sure that your sample buffer does not have too much salt in it.
(3) Gel imperfections – especially the separating gel surface (when allowed to dry before pouring the stacking gel) can also lead to this kind of issues. If using readymade gels, just make sure that the gels are well within the expiry date and were stored at recommended temperature all the time.
@Subhash, thanks for your reply!
1) What if the samples are too dense? Diluition should solve the problem?
2) It was standard RIPA buffer: never had any issues with it in the past, so I'd say it's unlikely. But in case there really is too much salt, do you have any suggestion on how to solve the issue?
3) The gels were precast, and always stored at the right temperature.
When you make protein extracts from different tissues, you should first normalise them by total protein concentration (before adding the sample buffer). Invariantly, when using manual grinding, or even freezing/thawing, the extraction efficiency is not the same, so you need to quantify total protein levels in your various samples. Afterwards, you bring all concentrations down to the lowest in your series by appropriate dilutions. Then you add equal volume of diluted samples and sample buffer, boil and run the SDS gel for later Western analysis. This means, when you run the SDS PAGE, you load equal voilumes and equal protein concentrations in each lane. If you just measure protein concentrations, and load different volumes to achive the same amount of protein in each lane, the samples will not run well. Always equal volumes and equal protein levels, it's more work, but it is necessary.
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