The datasheet gives us valuable instruction which is very important to complete our experiment successfully. But, sometimes datasheet does not provide everything, for example, primary antibody dilution range. In this case, how can I optimize to carry over the process? Have any alternative method exist to overcome this problem?
Antibodies are often diluted 1,000 - 10,000x, but this depends very much on the antibody and the concentration it's provided in. Check to make sure the product isn't labeled as 1000x or something similar. If not, I'd highly recommend contacting the supplier, since antibodies are often quite expensive and your target may also be costly or tedious to generate.
Which applicaiton are you using it for? This is important: for western blot antibodies may be more diluted than for example for immunofluorescence, so it certainly depends on your application.
If you are doing western and you are not sure, start with 1:1000 for the primary. The seconday can always be twice diluted as the primary was so 1:2000 in this case.
If you are doing Immunofluorescence and you are not sure, start with 1:200-1:500 for the primary depending on how valuable your antibody is. The seconday can always be twice as diluted as the primary.
If you are doing flow cytometry, use 5ul of antibody per ml of final solution and this should work.
It depend of your antibody quality. but in general in primary antibodies the starting dilution is 1-5 ug / mL for immunohistochemistry and Immunofluorescence.
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