Can results obtained in Western blot be representative for other techniques?

One antibody technique – one validation

When working with an antibody that was recently produced, and not yet used in different techniques, it is of crucial importance to confirm that the antibody in question binds to the target protein it was produced to. Different techniques can be used to achieve this, and one of them is Western blot. In this technique, both wild type extract, and extracts from specific knockdown or knockout mutants of the target protein should be included. The obtained results are not representative of using the given antibody in any other technique, as each technique requires a separate validation.

Antibodies bind to specific epitopes on the target protein, and these can be differently exposed depending on how the sample is processed. In Western blot (WB), i.e. denatured conditions, the antibodies will bind to linear epitopes, while in immunoprecipiatiopn (IP), antibodies binding to conformational epitopes, brought by protein folds, will occur.

Each technique has a specific antibody validation method.

Can results obtained in Western blot be representative for other techniques?

One antibody technique – one validation

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