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Normalization of Western blot data is an analytical step that is performed to compare the relative abundance of a specific protein across the lanes of a blot or gel under diverse experimental treatments, or across tissues or developmental stages.
The key criterion for the use of a housekeeping gene in this manner is that the chosen housekeeping gene is uniformly expressed with low variance under both control and experimental conditions. Validation of housekeeping genes should be performed before their use in gene expression experiments such as RT-PCR.
Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for qPCR.
Western blot workflow. The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. [1]
The following represent genes that should probably not be used for reference purposes: GUSB, [5] RPLP0, [5] TFRC, [5] GAPDH, HSP90, and β-actin. Although they were once considered as "housekeeping genes," recent data suggests that they are not as reliable as once thought. [1] [2]
A housekeeping gene is a gene that is required to maintain basic cellular function and so is typically expressed in all cell types of an organism. Examples include actin , GAPDH and ubiquitin . Some housekeeping genes are transcribed at a relatively constant rate and these genes can be used as a reference point in experiments to measure the ...
The housekeeping gene clusters are required in all bacteria and are often major players in the main metabolic pathways including central carbon metabolism and amino acid synthesis. The gut-specific functions include adhesion to host proteins and the harvesting of sugars from globoseries glycolipids.
Immunoprecipitation of intact protein complexes (i.e. antigen along with any proteins or ligands that are bound to it) is known as co-immunoprecipitation (Co-IP). Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins.