For example, an antibody that recognizes the protein only in its native form should not be used on samples using denaturing conditions, such as western blot. It is therefore essential to prepare the test samples accordingly. RNA levels are often very unreliable in determining the quantity of proteins.įurthermore, antibodies may or may not recognize the protein in its native or denatured state.It can be challenging to find negative cell lines or tissues, particularly for essential housekeeping genes.Expression data may not be complete in some cases.However, there are a few limitations to defining protein expression profiles using online databases: You can find information about target protein expression in different tissues or cell lines in peer-reviewed papers and online protein databases, including: It's often challenging to determine cell or tissue types that do or do not express the target protein. When a true negative control is not available, a sample expressing low levels of the target proteins can work as an acceptable alternative. A negative control is a cell line or tissue sample that does not express the target protein and, therefore, can provide the data on the non-selective binding properties of your antibody.A positive control is a relevant cell line or tissue sample strongly expressing the target protein of interest that can be used to confirm the selective binding of your antibody.Identifying and using appropriate positive and negative controls is essential for successful antibody validation. Choice and preparation of positive and negative controls Key points to consider when validating antibody specificityġ. Therefore, your antibody validation steps are essential because they are specific to your setup. Although a good manufacturer usually tests an antibody in several applications and species, it's impossible to account for numerous protocols and reagents with which researchers may use the antibody. Here we will focus on how you can validate antibody specificity in your experimental setup to ensure accurate and consistent results. Reproducibility- showing that your validation data can be reproduced in any lab.Affinity - showing the strength of binding between antibody and epitope.Specificity and functionality - showing an antibody can differentiate between various antigens in the intended application.Antibody validation revolves around proving three key aspects:
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