In accordance with recommendations by the International Working Group for Antibody Validation (Uhlen et al. 2016), we use various methods to validate PrecisionAb Antibodies. Our methods include immunoprecipitation-mass spectrometry (IP-MS) antibody validation.
To achieve IP-MS validation, first we demonstrate the antibody can be used to immuno-precipitate its target from cell lysates (Figure 1 and Figure 2). Following IP, we analyze the pulled down protein using a mass spectrometer, whereby sequences from the digested protein are identified.
In order to verify target specificity, after subtracting the background, we look at the number of unique peptides detected and their protein abundance.
Fig. 1. IP-MS antibody validation workflow. Incubate the cell lysate with the antibody of choice. Following the IP of the target, carry out the elution step. Resolve the samples by gel electrophoresis and analyze through proteomics. Significant proteins are identified by a high number of unique peptides and a high abundance.
Fig. 2. Immunoprecipitation of CD44 from HeLa whole cell lysate. The input lane is 10% input; the CD44-IP lane is the immunoprecipitation with the CD44 Antibody (2.5 µg, MCA2726); the IgG-IP lane is the immunoprecipitation with Mouse Monoclonal IgG (2.5 µg, MCA929). Western blot analysis was performed using CD44 Antibody (1/1,000, MCA2726) followed by detection with HRP Conjugated Immun-Star Goat Anti-Mouse IgG (1/10,000, 1705047) and visualized on the ChemiDoc MP Imaging System. Arrow points to CD44.
The antibodies that have been IP-MS validated are marked with an IP-MS flag and a PrecisionAb enhanced validation logo on product webpages.
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Learn about the validation methods implemented by Bio-Rad, including CRISPR-Cas9 gene editing, siRNA, and immunoprecipitation followed by mass spectrometry, ensuring the specificity and reliability of our antibodies in your application.
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