western blot chemiluminescence detection

In the enhanced validation data presented for the antibodies, the Western blot lanes in the high and low cell lines are displayed together with their corresponding RNA values. The cell lines are selected to express at least a five-fold difference between the RNA expression in the high and low samples. Antibody specificity is confirmed when the antibody signal matches RNA levels in the evaluated samples.įor each antibody, two tissues or endogenous cell lines are chosen for the validation: one with high RNA expression and the other with low or no RNA expression of the target protein. The orthogonal validation method validates the antibody staining using a non-antibody-based method.įor example, the Western blot results are compared with RNA-Seq data for the same samples, using both positive and negative controls. Orthogonal validation by comparing antibody Signal in Western blot to RNA Recombinant Expression Validation (validating with an over-expressed version of the target protein.).Independent Antibody Validation (comparing two or more antibodies targeting different regions of the same protein).Genetic validation (downregulation of the target gene).Orthogonal validation (verifying with a method other than antibodies).

In Western blot, four different enhanced validation methods are applied: To further demonstrate specificity, the validation performed for our antibodies is expanded with application-specific Enhanced Validation. The peroxidase (HRP) labeled antibody is visualized by chemiluminescence detection using a CCD-camera system. The endogenous protein lysates from mouse and rat cell lines are tested for many antibodies, When suitable cell lines are not available, recombinantly produced full-length target proteins in the form of HEK-293 cell line over-expression lysates are used as positive control samples. Protein lysates are selected based on RNA expression levels and the scientific relevance of the target. Factors that can affect proteins migration, such as alternative isoforms and post-translational modifications are considered,Įndogenous protein lysates from human tissues and cell lines are primarily used as samples. Our antibodies, Triple A Polyclonals, and PrecisA Monoclonals are routinely validated in Western blot.Ĭorrect binding is verified by comparing the band size with the theoretical mass of the target protein. Go to protocols Validation in Western blot Looking for a different protocol? See all our protocols for IHC, WB and ICC.

Western blot chemiluminescence detection