Hi all,
I have to do immunoblot with gastric fluid which are very acidic.
I've tried without any modificiation first, then...
- the gastric fluid was not mixed with 5x loading buffer,
- some particle was made after mixing with loading buffer,
- and finally, the blot was messy.
(c.f. The volume of gastric fluid is just around 50~300 ul, and the target molecule to be measured is not much to detect.)
I've searched for some specific protocol but couldn't find a good one.
If you have seen any paper/protocol for immunoblot with acidic sample, please let me know.
- Thank you.
Hi, if you are having problems with the pH of the sample then I would suggest buffer exchanging (desalting) the sample into a buffer closer to neutral pH such as PBS. You could do this by dialysis or using commercially available desalting columns (e.g. NAP-5 columns). If the sample pH is the issue with your blots, that should help.
Hi,
I've personally tried blotting a very acidic sample successfully with 1x loading buffer and %12.5 SDS page (Sambrook and Russell protocol). The colour of the sample gets yellow when you mix it with the loading buffer but as soon as you load it to the gel and run it for a few minuets the colour will turn back to the normal blue. However, as Mark said my problem was solved by extensive dialysis against PBS.
Hope it helps.
An alternative to the above would be a CHCl3/MeOH precipitation of your samples (10 volumes, 1:1). This would both remove the acid and concentrate your samples to help with detection. A warning, though, some proteins are soluble in acidic organic solvents.
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