For the western Blot, I need to check another antibody in my membrane but this antibody is from the same species as the first antibody so how can I do that without stripping? (Note: two antibodies are around 15 KDa away from each other.)
If you are avoiding stripping, then you have do it from the antibody angle. You can find one antibody for IgG2a and the other antibody for IgG2b, and with secondary antibody from different host, you are now able to probe both your antibody 1 and 2 with same species origin.
Thanks, Yi-Chun Cheng , but do you mean that I should use two secondary antibodies from two different hosts?
Hi Fatma Mohamed Fouad
In case you have enough sample volume and enough empty lanes in the gel, load again the samples leaving a space (one lane loaded with a marker). after blocking you can cut the membrane vertically and incubate it with the primary of interest.
Another suggestion (with taking risk in your consideration) based on proteins MW. If the proteins have HMW, run them on 6% gel, this would widen the distance between the two proteins. Then, you can cut the membrane. Again it is risky.
Fatma Mohamed Fouad Yes, when using primary antibodies with different IgG subclasses eg. IgG2a & IgG2b, you can choose donkey-anti-species IgG2a for the first target, and goat-anti-species IgG2b, as such.
If you are probeing with multicolor (like Li-COR) then you can apply the antibodies at the same time, which is highly recommended. If not, then you can probe the first target then shift to the next.
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