What is a loading control in PCR?
Beta-actin, also known as a “housekeeping” protein, is usually used as a loading control , for among others, the integrity of cells, protein degradation, in PCR and Western blotting. They can be used to normalize the levels of protein detected by confirming that protein loading is the same across the gel.
What are loading controls in western blots?
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.
What does a loading control do?
A loading control is a protein used as a control in a Western blotting experiment. They are used to make sure that the protein has been loaded equally across all wells.
Why is beta-actin a loading control?
Beta-Actin (42 kDa) is commonly chosen as a loading control due to its general expression across all eukaryotic cell types. The expression levels of this protein do not vary drastically due to cellular treatment, which is another reason the protein makes a suitable control.
What is a loading control SDS PAGE?
Loading control antibodies are used to ensure that equal amounts of each protein sample are loaded into the wells of an SDS-PAGE gel. Selecting a proper loading control whose signal remains constant under the tested conditions is essential in order to properly interpret the results of the western blot experiment.
Why we use loading control in Western blot?
Loading controls are antibodies that are used to detect proteins within samples. When western blots are used to determine the levels of protein expression in a sample, loading controls ensure that the results aren’t due to loading or protein transfer errors.
What is a good loading control for Western blot?
Therefore GAPDH, together with beta actin and tubulin is one of the most commonly used loading controls. We offer alpha tubulin and GAPDH DyLight® 680, DyLight® 750 and DyLight® 800 conjugated antibodies which are perfect as loading controls in fluorescent Western blotting experiments.
Is vinculin a housekeeping gene?
For high molecular weight (over 100 kDa) proteins we recommend Vinculin as a loading control. GAPDH is listed as a Top 20 housekeeping gene with the highest and most consistent average expression (She et al., 2009).
How many kDa is Gapdh?
146 kDa
GAPDH is a 146 kDa tetramer composed of four 30-40 kDa subunits.
What is the role of B actin?
This is one of the two nonmuscle cytoskeletal actins. Actins are highly conserved proteins that are involved in cell motility, structure and integrity. Alpha actins are a major constituent of the contractile apparatus.
What do you need to know about loading controls?
Loading controls are needed for proper interpretation of western blots. Find the right one for your experiment. Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample you are studying. Housekeeping genes are often chosen for use this purpose.
How are loading controls chosen for a protein?
Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample you are studying. Housekeeping genes are often chosen for use this purpose. Select a loading control that has a different molecular weight to the protein of interest.
Why are housekeeping genes chosen for loading control?
Housekeeping genes are often chosen for use this purpose. Loading controls are absolutely essential to ensure the reliability of your data when comparing expression of a protein in different samples. Why are loading controls essential? Why choose our loading control antibodies?