In western blotting I confirmed that the proteins were transferred from the gel to the membrane and was sure that the antibody was functional, but then there is no color formation when adding the substrate (DAB). Is there any way to check the activity of DAB? I am using Sigma antibodies and the secondary antibody is HRP conjugated. I have attached the protocol which I followed. I am not using any particular kit.
Marugadas, the chemiluminescent method is easier than colorimetric revelation method. You need to standardize the DAB color formation. To check if DAB is working, you need adjust the secondary antibody (decrease the dilution) and you need to take a good recipe of DAB (H2O2), so you start to changing the DAB recipes and 2º antibody. Good work, don't worry, it's not easy...
Can i have the working protocol or reference for chemiluminescent method? Do we need any speciall kind of instrument for this method?
Murugadas,
chemiluminescent method is so easy, is the same you are doing, but in the end you need to employ the chemiluminescent revelator, but you need a specific instrument to read (equipment revealing chemiluminescence) or by raio-X.
Search by papers that use this methodology, is so common.
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