Perform titration experiments on your samples to determine the concentration of antibody against your housekeeping protein and blot exposure time that gives optimum signal-to-background ratio without signal saturation. Probing with a loading control can be carried out alongside the target antibody by cutting the membrane before antibody incubation, or after the blot has been stripped of previously bound antibodies. Don’t use a housekeeping gene that you’ve found to vary in expression level during your experimental treatment previously. This allows you to easily distinguish bands representing your protein of interest from those representing your housekeeping protein.Įnsure your loading control is highly expressed in your sample and its level remains unchanged throughout an experiment. Journals now require appropriate controls to be used in your experimental setupĮnsure your protein of interest and loading control have different molecular weights. They ensure you have loaded and transferred equal amounts of protein across all wells of your western blot Loading controls are particularly necessary when signal levels of modified proteins are compared between samples
GOOD LOADING CONTROL WESTERN BLOT SOFTWARE
Tools for normalizing gel loading differences and western blot quantification (by software programs such as ImageJ) Expression levels of some housekeeping proteins may vary with cell type and treatment (Hagner-McWhirter 2015) *The exact molecular weight might vary from species to speciesĪntibodies that detect a highly expressed protein in a certain cell or sample typeĮncoded by housekeeping genes, housekeeping proteins are proteins required for the maintenance of basic cell functions and are often ubiquitously and abundantly expressed. Click on your housekeeping protein of interest to find out more information on the antibodies that Bio-Rad offers and select the housekeeping protein antibody that is right for your experiment. Table 1 shows the molecular weight and cellular localization of the commonly used housekeeping proteins. This is where western blotting loading controls can help.
However, to draw conclusions from a western blot, you need to ensure that the observed differences are only due to altered protein expression levels rather than gel loading or protein transfer errors. Generate Publication Quality Western blotsĪ western blot provides a readout of differences in protein expression levels, for example changes in protein levels over a certain time period, differences in knock-out versus wild type cell lines, and altered expression in patient samples.
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