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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.
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.
Although the terms "housekeeping gene" and "reference gene" are used somewhat interchangeably, caution must be used in selecting genes for reference purposes. Suitable reference genes must be stably expressed in the specific experimental condition or tissue of interest. [ 1 ]
Actin beta is often used in Western blotting as a loading control, to normalize total protein amounts and check for eventual protein degradation in the samples. Its transcript is also commonly used as a housekeeping gene standard in qPCR. Its molecular weight is approximately 42 kDa. [citation needed]
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]
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 ...
Thus both the number and type of housekeeping genes interrogated by MLST may differ from species to species. For each of these housekeeping genes, the different sequences are assigned as alleles and the alleles at the loci provide an allelic profile. A series of profiles can then be the identification marker for strain typing.
Calnexin (CNX) is a 67kDa integral protein (that appears variously as a 90kDa, 80kDa, or 75kDa band on western blotting depending on the source of the antibody) of the endoplasmic reticulum (ER). It consists of a large (50 kDa) N-terminal calcium-binding lumenal domain, a single transmembrane helix and a short (90 residues), acidic cytoplasmic ...