Hi everybody,
I am trying to block Fc receptor by human IgG for a specific experiment (it's not flow cytometry), in an adherent cell, not a suspension. I know my primary antibody working concentration but I don't know how to calculate how much IgG I need for blocking? Is there any ratio between the first antibody and IgG concentration?
Your help is highly appreciated.
Hello,
I do not have an exact answer to your question, but i can offer my little piece of experience. Maybe it helps you find your answer.
Firstly, doesn't your provider give you the protocol for Fc-blocking? They usually do.
It pretty much depends on what cells you are using. I will give this one example and you can search for a better one afterwards.
https://www.biolegend.com/en-us/products/purified-anti-mouse-cd16-32-antibody-190
This is anti-CD16/CD32 which is used especially for B-cells, but also for NK, Monocytes/Macrophages, etc.
You have all the info in there: the concentration is 0.5mg/mL and the producer suggests < 1ug/10^6cells/100uL.
Maybe this will give you an idea of the dimension of the concentration you seek?
Usually, it is 200 ug/ml for whole IgG, which is approximately 100-200 fold higher than for antibody against a specific antigen.
However, it is dependent on cell types, concentration of specific antibody and experimental design:)
You are more safe with non-immune cells in the relation to non-specific binding.
However, if you're going to do experiment with human monocytes, then titration of both non-specific IgG's and specific IgG(s) (your antibody) is highly recommended.
Sergey Ryzhov Thanks for the reply, I just had a run with 100 times non-specific IgG at the same time with my specific IgG. Actually, I am not using immune cells, they are cells treated with radiation which have a lot of inflammatory responses. I wanted to block other non-specific FC receptors on the cell surface. It seems that with this concentration, my specific IgG does not show any binding at all. Do you think titration will work for my experiments?
100-fold excess is a general approach, widely used to distinguish between specific and non-specific effects. If you don't see binding of your specific IgG's with 100-fold excess of non-specific IgG's, then most likely this is a non-specific effect. It is hard to suggest anything else in the absence of specific information such as method you are using to measure IgG's binding, cell type, and information on your specific and non-specific IgG's.
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