The selection of an appropriate isotype control is key for flow cytometry experiments. Isotype control antibodies that have no relevant specificity can distinguish non-specific "background" staining from specific antibody staining. Three main factors contribute to the levels of background staining associated with a primary antibody: a) binding to Fc receptors on target cells, b) non-specific protein interactions with cellular proteins, lipids, or carbohydrates, and c) cell autofluorescence. All of these factors vary greatly depending on the target cell type and the isotype of the primary antibody. Therefore, in order to accurately determine the level of specific staining by the primary antibody, isotype controls need to be matched to the specific primary antibodies being used. GenScript offers a series of isotype control antibodies that can be used in your research.
Loading Control Antibodies
Loading control proteins are known to be constitutively and stably expressed at high levels in almost all tissues and cells. Housekeeping proteins, such as GAPDH, tubulin, and actin, represent some of the well-known loading control proteins.
Loading control antibodies are used in Western blotting for two main purposes:
To estimate relative amounts of total protein loaded in each lane. (This information is needed to determine if the difference in levels of the protein of interest in different samples is due to loading and transfer errors or to real changes in protein expression.)
To troubleshoot the Western blotting procedure.
GenScript offers a wide selection of polyclonal and monoclonal loading control antibodies for GAPDH, α-tubulin, and β-actin for several species. HRP- and biotin-conjugated versions of these antibodies are also available.
Primary Antibody: A. 0.1 μg/ml THE™ β-Actin mAb(Mouse)(GenScript, A00702) B. 0.1 μg/ml β-Actin mAb (Mouse) (Company X) Secondary Antibody: Goat Anti-Mouse IgG (H&L) [HRP] pAb (GenScript, 1:10,000, A00160) The signal was developed with Lumisensor™ HRP Substrate Kit (GenScript, L00221V500)
Primary Antibody: 0.1 μg/ml GAPDH Antibody, pAb, Goat (GenScript, A00191) Lane 1-8: 5.0, 2.5,1.25, 0.62, 0.31, 0.16, 0.08 and 0.04 μg of Hela cell lysate The signal was developed with One-Step Western™ Fluorescent Kit (GenScript, L00397)