| Antibodies are used in a variety of applications and contribute greatly to scientific research. However, poorly validated antibodies is a reason for the ongoing reproducibility crisis. Therefore, it should always be confirmed that the produced antibody (custom-made or catalogue antibody) binds to the desired target protein. This can be achieved by: Direct approaches • Reliable knockout/knockdown mutant for a target protein are used • Immunoprecipitation and MS of its product, which confirms the protein identity (IP-MS) Indirect approaches • Antibody is confirmed to recognize recombinant antigen • Band of the same MW as the target protein is not present in pre-immune sample • Competition assay (neutralization assay) where antibodies are saturated using the antigen (free peptide or protein) • Target protein is confirmed to migrate at the correct MW (processing sites and modifications can affect apparent MW) • Antibody has low cross-reactivity • Samples from specific conditions, up or down regulating expression of target protein are used • Same band is recognized by two independent antibodies to the same target protein • Target protein band is recognized in multiple species An orthogonal approach is a combination of direct and indirect approaches. Trusted antibodies should be evaluated using combined approaches. Validation has to be conducted separately for each technique. Antibodies validated in Western blot, require proper validation for immunolocalization studies, immunoprecipitation and other techniques. | Check Agrisera Western Blot Guide Request your free copy today! In case of any questions, you are always welcome to contact us Each antibody-antigen interaction is unique and presents different challenges in terms of antibody specificity, sensitivity and functionality. This has to be taken into account during antibody validation process. |
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Why, to detect some proteins, 6M urea must be included in the extraction and loading buffers?2023-09-01 I have not used this antibody before, what conditions of Western blot do you recommend?
2023-07-27 Which subclasses of immunoglobulin G are found in rabbit serum?
2023-07-18 Antibody suddenly does not work any longer, what can be the reason?
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2023-05-10 What is a reason for so called "ghost bands" to appear on blots?
2023-03-30 One of the ways to remove background on a Western blot
2023-03-24 How to predict in which technique a given antibody may work?
2023-03-10 Which capture antibody to use in ELISA?
2023-03-06 Why should we measure protein concentration?
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