Greetings
I am doing WB, and have problems stripping pERK in particular, but also ERK, from the membrane. I am using this product to strip it with:
https://www.thermofisher.com/order/catalog/product/21059
I am using 15 ml stripping solution per membrane, incubating for 10 minutes at 37 C. Then washing 3 x 10 minutes with PBS + Tween, then blocking with
https://www.thermofisher.com/order/catalog/product/37538 for 30 minutes, and then incubating secondary Ab again to see if the previous antibodies have been stripped. And they are not stripped by this process. I have also tried using a "plus" version of the stripping buffer, but to no avail. Someone from my lab said it is almost impossible to strip pERK, but I have done so previously with the same protocol, so I dont know what could be wrong.
Any experiences?
Thanks in advance
We did stripping for pErk to probe total Erk. But we followed the protocol of Abcam (Stripping buffer composition for mild stripping ) and it worked.
I always probe pERK(1/2) at 4 degree O/N. You may try to strip it for a longer time. 1 hr at RT will also work.
Hi,
If you are using HRP antibodies, you can bleach the membrane with 30% H2O2 for 20 minutes. We do this for our streptavidin blots since the association is so strong between biotin and streptavidin, but this will work for any HRP-conjugated antibody to completely remove the signal. However, the primary antibodies should still be different species. You won't need to block again after bleaching, although you will definitely want to do a couple washes to remove residual H2O2. If you want to be extra thorough, you can put ECL on the blot and image real quick to make sure the blot is fully bleached, but I've never had a problem. And even if your first antibody binds to a specific protein, after bleaching you can probe with another antibody against a different epitope of the same protein and you should be able to see signal.
Hope this helps!
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