![]() While the western blot is often considered semi-quantitative or qualitative, it can be quantitative with infrared fluorescence-based detection 13, 14, 15, 16, 17, 18. One major application of antibodies in both large and small labs is the western blot. Antibody validity is highly dependent on biological context and the assay itself 10, 11, 12. For example, qualitative inference from immunohistochemistry may be possible, but acquiring quantitative data from flow cytometry may not be with the same antibody and cell system. The intended application for the antibody brings potentially different criteria and stringency for their use. Antibodies are widely-used critical tools in a variety of biomedical research assays, but they are not always suitable for the application of interest. While there are many potential reasons for lack of reproducibility, one major reason relates to research reagents, including antibodies 10, 11, 12. Scientific research, and in particular that in the biomedical field, has come under harsh scrutiny and debate of late due to questions of reproducibility 1, 2, 3, 4, 5, 6, 7, 8, 9. Importantly, we demonstrate that results from microwestern analyses scale to normal “macro” western for a subset of antibodies. Total antibodies generally required higher working concentrations, but more comprehensive antibody panels are required to better establish whether this trend is general or not. Phospho-specific and total antibodies do not have discernable trend differences in linear range or limit of detection. Linear range for all validated antibodies is at least 8-fold, and up to two orders of magnitude. In some cases microwestern struggled with higher molecular weight membrane proteins, so the technique may not be uniformly applicable to all validation tasks. Pilot experiments demonstrate a high proportion of investigated antibodies (17/24) are suitable for quantitative use however this sample of antibodies is not yet comprehensive across companies, molecular weights, and other important antibody properties, so the ubiquity of this property cannot yet be determined. Here, we use microwestern array to rapidly evaluate suitable conditions for quantitative western blotting, with up to 192 antibody/dilution/replicate combinations on a single standard size gel with a seven-point, two-fold lysate dilution series (~100-fold range). Note : For murine tissue samples, conjugated mouse antibody HRP-60004 and rabbit antibody 10494-1-AP are preferable.Fluorescence-based western blots are quantitative in principal, but require determining linear range for each antibody. GAPDH interacts with other proteins to regulate nuclear translocation and GAPDH catalytic activity. Some of What are the known protein-protein interactions of GAPDH? Useful as a control for Western blots and RT-PCR. Yes! Because GAPDH is often highly expressed throughout different tissues and cell types, GAPDH expression is Is there a reason to use an antibody targeting GAPDH if my research is not concerned with glycolysis or nuclear GAPDH is expressed primarily in the cytosolic and membrane regions, with minimal expression in the nucleus Is a key factor in regulating apoptosis, translocating to the nucleus under certain stressful conditions and actingĪs a signaling factor in oxidative stress-induced apoptosis. Other studies indicate that GAPDH plays a role in GAPDH is responsible for catalyzing the phosphorylation of glyceraldehyde-3-phosphate into D-glycerate-1,3-biphosphate. While this function of GAPDH is considered its primary function, GAPDH is also involved, evenĬritical, in other cell functions, some of which are still being studied. Recent studies have suggested that GAPDH It is recognized as an important enzyme involved in metabolic pathways, aiding in the glycolytic GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase and is often referred to as a "housekeeping"
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