What is the purpose of using control proteins such as actin or GAPDH?
Beta-Actin Protein Loading controls are essential for proper interpretation of western blots. They can be used to normalize the levels of protein detected by confirming that protein loading is the same across the gel.
Is GAPDH a good loading control?
GAPDH (36 kDa) is integral for glycolysis and plays many roles in nuclear function; such as transcription regulation and apoptosis. The stable and ubiquitous expression of GAPDH also make it a suitable loading control for many experiments.
What type of protein is GAPDH?
GAPDH is a homo tetramer containing four identical 37 kDa subunits. Localized in chromosome 12, human GAPDH gene transcribes single mRNA species, consequently producing a subunit that comprises a polypeptide chain of 335 amino acids7,8.
Why is GAPDH used as a control in PCR?
As one of the most common reference genes, GAPDH is often used to normalize the gene expression data, being used as an endogenous control in the quantitative analysis of RT-PCR, since in some experimental systems, its expression is very constant (Edwards and Denhardt 1985; Winer et al. 1999).
What is the purpose of a loading control?
Loading control antibodies are important controls as they indicate the equal loading of samples across all wells. Loading controls also indicate the proper transfer of proteins to the membrane during the western blotting process. Loading controls are typically proteins with high and ubiquitous expression.
Is GAPDH a good housekeeping gene?
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a well-known housekeeping gene with functions in glycolysis. Many biologists are familiar with the gene and use GAPDH antibodies for a loading control when performing western blots.
Why is GAPDH used as a reference gene?
In bovine oocytes, GAPDH was selected as reference gene because its expression showed stability (Van Tol et al. 2007); in zebrafish embryos, the GAPDH gene was considered less stable (Lin et al. 2009).
Is GAPDH part of glycolysis?
GAPDH is the sixth enzyme of glycolysis, whereas CytOx is the fourth complex of the mitochondrial oxidative phosphorylation system. Both GAPDH and CytOx are highly regulated under normal energy metabolic conditions, but both of these enzymes are highly deregulated in the presence of oxidative stress.
Is GAPDH a protein?
GAPDH, a multifunctional protein, not only catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate during glycolysis, but also regulates multiple cellular functions including membrane fusion, transport, apoptosis, DNA replication and repair, and regulation of transcription and translation.
What type of protein is glyceraldehyde 3-phosphate dehydrogenase?
Glyceraldehyde 3-phosphate dehydrogenase (abbreviated GAPDH) (EC 1.2. 1.12) is an enzyme of about 37kDa that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules.
Why is GAPDH used as a housekeeping gene?
GAPDH is a key regulatory enzyme, which catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate during glycolysis. β-actin is a cytoskeletal protein involved in cell structure and motility. Based on these basic and ubiquitous cellular functions, GAPDH and β-actin are considered as housekeeping genes.
Can a GAPDH protein be used as an internal control?
Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples.
Why are beta actin and GAPDH important loading controls?
When considering a loading control for your experiment, it is important to choose a protein that does not have a similar molecular weight to your target of interest. Additionally, the loading control protein should demonstrate ubiquitous expression. The most popular loading control targets include Beta-Actin and GAPDH.
Why is GAPDH important as a loading control?
Novus offers a complete selection of highly characterized beta-Actin antibodies. GAPDH (36 kDa) is integral for glycolysis and plays many roles in nuclear function; such as transcription regulation and apoptosis. The stable and ubiquitous expression of GAPDH also make it a suitable loading control for many experiments.
How is GAPDH used in western blot analysis?
Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright Â© 2012.