1:5000

Hello Rana Abdoun

You will have to determine the optimal antibody concentration to achieve the best possible results. This may be achieved via two methods. The most efficient and affordable option is to use the dot blot technique on a nitrocellulose membrane. The other option would be to perform antibody titration on a PVDF membrane.

Proteins could be adhered to a nitrocellulose membrane (via dot blot) or PVDF membrane (via gel electrophoresis and transfer chamber) and then you may cut into strips that are used to screen combinations of serial dilutions of the primary antibody. For comparable results, it is essential to keep all protocol parameters the same namely, the sample type, time of incubation, number of washings and duration as well as temperatures. Also, the protein sample should contain an abundance of the antigen of interest. But many a times, the antigen concentration of the sample is not known. As a result you may have to use a wide range of dilutions for testing.

You may test a series of antibody dilution 1:250, 1:500, 1:1000, 1:2000, 1:4000 and 1:5000. Try to include a negative control sample with no primary or secondary antibody to reveal non-specific cross-reactivity. In this way, you will be able to optimize the ideal antibody concentration necessary to achieve the best possible results.

At times, the recommended antibody dilution mentioned in the datasheet may not work. Therefore, titrating antibody concentrations by using two-fold dilutions either side of the manufacturer's recommendations to identify the optimum antibody concentration for Western Blot being performed may help.

Best.

In case of using a new antibody or looking for a niche to buy you can visit a website called antibody pedia. You can find a feedback and some recommendation on the antibody of interest.