1: I am working with blotting p44/42 and measuring phosphorylation. After having stripped my membrane with "Restore™ Western Blot Stripping Buffer", I experience a white, very finely grained, pricipitate in the container keeping the membrane and stripping buffer. This precipitate is also found in the primary and secondary antibodies afterwards, even though I have washed thoroughly with PBS (-mg -ca) in between different steps. Is anyone else experiencing the same? Is it of concern? I am speculating that it might be actual protein from the membrane precipitating, which of course is of great concern, as it also "contaminates" my primary and secondary antibody stock then. I can provide more details if needed.
2: From the blot, I see third band of about 46 kDa. It is the upper band seen in the picture. I suspect that it might be ERK1b, but I would like to validate this. Does anyone else working with p44/p42 see the same band?
All experiments are carried out in GH3 being stimulated with ghrelin.
Thanks for any input and have a nice day.
As for the question 1, I think, you should definitely contact the customer support of the manufacturer. They are the best person to answer.
Besides, it might be worth investigating what the precipitates are made of.
If I were you, I would collect them, spin down the precipitate, and try to solubilise them in SDS-PAGE sample buffer, and if solubilised, load them on SDS-PAGE, to see if they are proteins.
It could come from the blocking reagent. If you know which proteins are in the blocking reagent, you may find them.
I agree that you should check your stripping solution before getting mad to understand what is going on. Of course your problem could be more easy to solve.
It happened to me once to use a not well clean bowl to ibridate a membrane with primary and secondary antibody; as a consequence I brought with me a 'precipitate' which contaminates both my stock. I figured out that such contaminant come from milk solution I used for blocking in a previous blot (as Shin-Ichiro suggested).
For the second question: which is the brand of your antibody? Which percentage is your gel?
Greetings guys. I just wanted thank you for replying, and get back to you, as some people might read this post and be curious as to what might have solved it. In this instance, it appears that the precipitate originates from the blocking buffer itself, and thus has nothing to do with loss of proteins or other bad stuff. I doubt it has any bad effects on the WB process either, but cannot confirm.
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