International Antibody Validation Working Group (International Working Group on Antibody Validation, IWGAV),On September 5, 2016, an independent group of international scientists with different research interests in protein biology announced online in the journal Nature Methods that they had developed a preliminary strategy to address the crucial unmet needs in antibody specificity, functionality, and reproducibility. The first proposal for validating antibody specificity strategies was published in Nature Methods (2016). Five "pillar strategies" have been identified (as follows) to guide antibody validation in specific applications.
Knockout / Knockdown,The abbreviation is KO/KD. KO/KD samples or cell lines are those in which the target gene has been knocked out (KO) using CRISPR/Cas9 technology, or knocked down (KD), meaning the expression of the target gene is reduced, through the use of siRNA/shRNA.
Protein Overexpression,The abbreviation is "OE." Overexpression (OE) of a target gene involves transfecting cells with a vector containing the target gene to increase its expression levels.
Immuno-Capture/Mass Spectrometry Analysis,The abbreviation is "IP/MS." Immunoprecipitation (IP) combined with Mass Spectrometry (MS), or IP-MS, involves using an antibody that specifically binds to the target protein to isolate it. This technique, when coupled with MS analysis, can identify the target protein that directly interacts with the purified antibody, as well as other proteins that may form a complex with the target protein.
Biological and orthogonal characteristics,The abbreviation is "Orthogonal" . Orthogonal approaches are based on biological characteristics and involve using non-antibody-dependent methods to quantify large numbers of samples, and to detect the correlation between antibody-based and non-antibody-based (e.g., RNA expression levels) quantifications. This includes altering antibody signals through changes in the biological properties of the target protein, such as natural differential expression, variations at the gene and protein level, metabolic changes, and alterations in signaling pathways. For example: 1. Natural differential controls: To validate the natural tissue or cell specificity of some target proteins, multiple groups of naturally differentially expressed cells or tissues can be used as controls. 2.Stimulation with pathway agonists and antagonists: Cells can be stimulated with pathway agonists and antagonists, causing changes in modification levels such as phosphorylation, acetylation, methylation, hydroxylation, and glutamylation.
Comparable antibodies,The abbreviation is "Comparable". Comparable approaches involve using two or more independent antibodies that recognize different epitopes on the surface of the target protein, and determining specificity through comparative or quantitative analysis.
Recombinant antibodies, The antibody genes are cloned into high-efficiency expression vectors in vitro. These vectors are then introduced into expression hosts to produce recombinant antibodies. Recombinantly produced antibodies overcome the limitations of other antibody production methods, providing you with the highest level of consistency between batches, confirmed specificity, and a guaranteed long-term supply.
Hot-selling products Typically, a target will have multiple antibody catalog numbers, which may differ based on the antibody's host species, clone number, and antigen recognition site. When the experiment does not specify requirements for the clone number or antigen recognition site, it is preferable to choose the best-selling catalog number.