Hello,
How reliable are western blots after they have been stripped? Does the buffer strip all lanes equally? If you load 20 ug onto a gel and strip twice, how much would be left?
I'm trying to optimize my western blots and I typically minimize stripping the gel to just once for the control antibody. I wanted to see other peoples' experiences.
Best,
I also load 20ug in my blots.
Personally, I strip three times very gently and my signals are very uniform. I notice that if I probe my blots out of sequence (going from, say, actin, strip, tubulin to tubulin, strip, actin) I can get high variation.
As long as it's on a shaker the stripping across lanes should be equal, since you should be putting on an ample amount of stripping buffer.
Hard to say how much protein is left since that depends so heavily on the efficiency of your transfer and stripping conditions. I've had some antibodies that are so well-made that trying to remove them nearly takes the ladder with.
I generally only strip 1 - 3 times. You can test how much of your protein will remain after a certain number of strips by blotting for the protein of interest, stripping the number of times you plan to use before measuring that protein, and blotting for that same protein and then comparing the signal between the two blots. I would also recommend testing your stripping method to make sure it works. After stripping, incubate with secondary antibody (skip the primary antibody incubation) and proceed as normal to make sure you cannot still visualize the protein after stripping. If the multiple proteins you are looking at are of similar MW, and the stripping protocol isn't effective, you will skew your results.
I am stripping 3 times with same as Nina Hahn but at 3rd time I got reduced band. So I am using 2 times with good protein band.
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