Hello everyone,
In terms of methods, should one increase concentration of antibody solution or increase the volume of incubation when the signal is weak? This could apply to the primary or secondary antibody. On a cell, there is a finite number of places where the primary antibody count bind, so there is theoretically a minimum number of antibody molecules needed per cell. If a condition in the experiment were to increase the rate of cell division over the course of the experiment, you potentially could have many more cells in one conditions over the other, and one signal may look weaker compared to the other if there isn't enough antibody to label the cells, correct?
Generally you can just decrease the dilution factor, effectively increasing your concentration of antibody.
If you're currently diluting at 1:500 and not seeing any signal, try 1:100.
It is less the amount of antibody that you add to a sample then the moleculardynamic movements and bindings that are based on the concentration. Therefore I would recommend, like Samantha and Mohamed mentioned, to decrease the dilution factor. And as Mohamed mentioned, you should always do several conditions. In the best case you titrate your 1AB against your 2AB. It takes a lot of time but specially if the antibody is new or you can't find a matching protocol for your experimentan onset it might be helpfull. Sometimes a simple change of washing buffer or blocking solutions could make the difference or a different fixiation method.
I found that a 1:200 dilution of my antibody worked well once before and found that it worked well for staining. When I performed that test, I stained about 75,000 cells in a gel. This time I am staining twice the number of cells. I am thinking of keeping the 1:200 dilution, but should I increase the incubation volume? What is a good rule of thumb when choosing an incubation volume?
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