Question: I have received an antibody which has not been used before in Western blot, what protocol should I use? To save time, before doing any experiments, it is recommended to conduct a short theoretical analysis and answer the following questions:
| Samples: WT, specific mutant Protein load/well: >50 µg Blocking: 5% non-fat milk, 1 h/RT Primary antibody: 1: 500 or 1: 1000, ON/4°C Secondary antibody: 1: 25 000, 1 h/RT Record results Including samples from knockout or knockdown mutants is crucial, if the antibody is used for the first time. This will confirm its binding to the correct target protein. Using other controls at this stage, as for example overexpressed protein or actin, is not relevant. Without a mutant sample, we will not be able to confirm tha the antibody is binding to the correct protein. If it is not possible to obtain vital mutants, there are indirect method to confirm binding of the antibody to the target protein under investigation. To avoid false positive results with antibodies, the used set of samples have to be chosen carefully. |
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