If I reprobe a membrane that I previously developed using anti-mouse (without stripping) with anti-rabbit, why do i still see the signal for the mouse antibody? My colleague in the lab does not have this problem and we use the same antibodies, dilutions and incubation times. I am trying to see two bands on one membrane that are of different sizes. I don't know why he does not see the old bands but I do.
Does your colleague strip before the second (anti-rabbit) probe? You normally have to strip before continuing with a new probe.
The enzyme-linked secondary antibodies usually are very specific, so an anti-rabbit antibody doesn't recognise mouse antibodies sitting on their blotted antigen. However, the enzyme on the anti-mouse secondary (i.e. HRP) is quite stable and depending on how harsh your stripping conditions, you may have some left that will give a signal after incubation with the rabbit serum (+ anti rabbit) to detect another antigen. If your colleague does not strip and gets away with it, it means that his first antigen gives a weak signal, and the second antigen gives a strong signal (which is how you should proceed). Even without stripping, the first enzyme will loose some of its activity and a weak signal will become even weaker. If the second antigen is more abundant, or the antibody is better, the signal will be much stronger, and if you expose for a shorter period at the second detection step, you don't see the signal from the first antibody pair bleeding through.
I always say, if you want to detect two different proteins, load a large gel with a set for one antibody, then a size marker, and then a set for the other antibody. Blot and cut the nitrocellulo0se in half in the middle of the size marker so that each blot gets a fair share of the marker and you can position things. Then develop each blot with their specific antibody and expose with optimum conditions. You cannot compare two different antisera anyway (apples and pears), and stripping is always risky as it can remove antigens from the nitrocellulose too. If your gels are not big enough to run two sets on the same tgel, buy bigger gel electrophoresis tanks.
The HRP on the secondary antibody will remain active. Your colleague probably kills the activity with sodium azide. Did he re-probe with a new primary antibody with NaN3 in the solution?
What Mark has mentioned above is a valid point. Any case, when I reprobe (without stripping), the residual signal varies from time to time depending on the initial signal strength. I usually make it a point to develop the weaker antibody first. Washing the blots immediately after first development also helps.
You should strip your membrane, otherwise you will cross-reaction between the two secondary antibody!
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