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#Purpose of western blot analysis serial

The signal intensity must correlate directly with the protein concentration within the range that will be used in the experiment.įigure 2.

#Purpose of western blot analysis serial

The dynamic detection range for a loading control can be determined by running a Western blot specifically for the protein control in serial dilution. That is, the protocol and detection method can reveal the changes in the levels of loading controls and the proteins of interest without signal saturation.

the detection limits for the loading controls and the proteins of interest are within dynamic ranges.

the detection bands of loading controls must not interfere with those of the protein(s) of interest (i.e., they should have substantially different molecular weights),.

the protein levels of loading controls remain constant (relative to the total protein content) under the test conditions (such as across treatments or developmental stages),.

The proteins serving as loading controls must fit certain criteria. The number of publications (num) that cites each type of main loading controls with antibodies from each of the major suppliers. Other interesting observations from this survey are discussed in this article later. The results indicate that actin (specifically beta actin) is the most commonly used control. Figure 1 displays the Western blot images compiled from some of those publications.

Labome has surveyed publications with loading controls in Western blots (Table 1). Looking to the literature can be very useful in considering potential loading controls for your Western blotting experiments. For this reason housekeeping genes that are constitutively expressed and necessary for basic cell functions, like cytoskeletal proteins, are typically used for loading controls. For any particular experiment it is important to consider potential loading controls and try to find a protein that is expressed at stable levels among the samples of interest. A variety of different proteins are used as loading controls. To use a loading control for these purposes detection with control protein antibody and the experimental antibody should be done on the same blot. The signals from loading controls are typically used to normalize the signals from the proteins of interest. - antibody incubation (for primary or secondary antibody), and signal detection across separate samples/lanes.- protein transfer efficiencies from the gel to the membrane among different samples/lanes,.- the amount of protein sample loaded in each lane,.Ī loading control accounts for potential variation in: