![]() Proliferating Cell Nuclear Antigen (PCNA) is a processivity factor for DNA polymerase δ it helps control eukaryotic DNA replication by increasing polymerase nucleotide processing ability during elongation of the leading strand. Proteintech’s COX4I1 antibody (11242-1-AP) was generated against a COX4I1 whole-protein antigen (amino acids 1–169) and also recognizes COX4I2. For an alternative mitochondrial protein loading control see our entry on VDAC1 below. It is also advisory to double check that any experimental manipulations do not affect its levels. However, some caution is advised when selecting this protein for Western blot detection as many other proteins run at its 17kDa size during SDS-PAGE (make sure your band of interest won’t be obscured). Because of its dependably high level, COX4I1 is commonly detected as an effective loading control for mitochondrial proteins. COX4I1 expression is ubiquitous throughout all tissues, whilst COX4I2 is lung-specific. The COX-4 subunit can be expressed as either of two isoforms, isoform 1 and 2 named COX4I1 and COX4I2 respectively. ![]() Actin antibodiesĬOX-4, or COXIV (cytochrome c oxidase subunit IV), is a nuclear-encoded subunit of the human mitochondrial respiratory chain enzyme cytochrome c oxidase (COX). However, if your studies involve work with skeletal muscle samples, or you are working with conditions that see changes in cell growth or altered interactions with the extracellular matrix, another loading control may be better suited to your needs. Many studies use β-actin antibodies recognizing all actin isoforms to probe for this loading control collective. Proteintech’s polyclonal ACTB antibody (20536-1-AP) was generated using a β-actin protein antigen (amino acids 14–167) and recognizes all forms of actin, making it a pan-actin antibody. HeLa cell lysate (10 ug/lane) was separated by SDS-PAGE and actin was detected by anti-ACTB antibody 20536-1-AP at varying dilutions. Western blot analysis of ACTB in multiple cell line and tissue lysates using anti-beta actin mouse monoclonal (60008-1-Ig) at a dilution of 1:5000. As such, actin is widely used as an internal control in Western blotting experiments Together, actins are the most abundant proteins in the typical eukaryotic cell, accounting for about 15 percent of total protein in some cell types. The beta (β) and gamma 1 and 2 (γ1 and γ2) actins co-exist in most cell types and are an integral part of the cytoskeleton they are mediators of cell trafficking, structural integrity and cell motility. The alpha actins - alpha C1 and alpha 1 and 2 - are a major constituent of the contractile apparatus in muscle tissues. The six isoforms of actin constitute a family of highly conserved globular proteins comprised of three main isoform groups, alpha, beta, and gamma. ![]() View our summary table below to help select a loading control for your sample type with a different molecular weight to your protein of interest. Here we’ve provided background information on each of the internal control proteins targeted by control antibodies we offer, helping you choose the best control for your personal needs… ![]() This means control candidates require careful selection even bastions of the loading control repertoire - such as β-actin and α-tubulin - can be affected by the conditions of your experiment (so be sure to double check your chosen manipulations do not impact them.) As mentioned above, the most basic criterion for a loading control is that its level remains unchanged throughout an experiment, regardless of tissues or cell types used and how they are handled. The loading control candidates for Western blotting are usually proteins with high and constitutive expression. This step is used to standardize results and normalize for any errors that creep into a Western blot experiment, such as sample loss through loading at SDS-PAGE or Western blot transfer. those with constant, unchanging levels - are usually detected in a second round of blotting, following primary detection of your protein of interest. Protein standards are required to make sense of Western blotting results, and check that any increases and decreases in target proteins are actually due to experimental manipulations and not, for example, because the sample went wandering during gel loading. Western blotting requires such controls: it is widely used for the semi-quantification of protein levels under of a set of different experimental parameters. ![]() The proteins and peptides that regularly serve as endogenous or internal controls may not always take center stage in your research, but they are indispensable to your conducting meaningful experiments and are essential for publication. ![]()
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