If you have or know any article or book or methods that can help me to do the dilution please attach it
I use this as a guide but as Md Riaj Mahamud says you need to try yourself with various dilutions and see which one works best.
https://www.abcam.com/protocols/antibody-dilutions-and-titer
Hi Suad Alghamdi,
I do not know any book or article to standardized the dilution of antibody for IHC or IF. But I can tell you what we practice in our lab. We usually dilute the antibody from 0.01 to 15ug/ml. We make different dilution of antibody in blocking buffer. Then use it for different slides to see which concentration has best signal compare to background staining. Of course you need to use positive control to optimize the antibody concentration before using in you experiment. Thank you.
I use this as a guide but as Md Riaj Mahamud says you need to try yourself with various dilutions and see which one works best.
https://www.abcam.com/protocols/antibody-dilutions-and-titer
- Antibodies validated for IF will say as much on the data sheet including dilution
- If you do not have you can easily obtain the pdf or no doubt dilution from either company web site or a general google query
- Generally, antibodies for IF are used at about 10 x concentration of what you use for Western blot
- Thus, if your data sheet makes ref to western blotting at 1:500 dilution then try at 1: 50 for IF
- Normally, in my experience antibodies for IF are used @ 1:10 (top end) to 1;100 corresponding to 1: 100 to 1: 1000 respectively by wetsern blotting
- If you are not sure pick a titrant - say 1: 20 and also try 1: 10 1: 50 and 1:100: Somewhere in that ball park in the absence of further info is likely to work
I think you mean the way to calculate the working dilution for an antibody. There is a simple ecuation (basic chemistry):
Initial dilution x Initial volume = Final dilution x Final volume
- Initial dilution: you must consider that the antibody is pure, so the initial dilution is 1.
- Final dilution: the working dilution is provided in the data sheet. For example, 1/50.
- Final volume: I adjust this volumen based on the number of sections that I have. I put 40-50 ul per sample. For example, if I have 10 samples, I have to prepare 500 ul of antibody (+ 50ul to be sure that I will have enough antibody).
- Initial volume: It's the volume that you have to take from the pure antibody.
Example:
1 x Initial volume = 1/50 x 500 ul
Initial volume = 10 ul of antibody
500 ul final volume - 10 ul antibody = 490 ul of serum (or any other blocking solution).
I hope to be helpful! :)
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