Why Does the Target Protein Show a Double Band in the Input but a Single Band in the IP?
In immunoprecipitation (IP) experiments, observing a double band for the target protein in the input sample but only a single band in the IP sample may be attributed to several factors:
Protein Modifications
The target protein in the input sample may exist in different post-translationally modified forms (e.g., phosphorylation, glycosylation), leading to variations in electrophoretic mobility and resulting in a double band. In the IP sample, the antibody might specifically recognize and precipitate only one form of the protein.
Protein Degradation
The double band in the input sample could be due to partial degradation of the protein. During the IP process, the antibody may selectively bind to and precipitate the intact, non-degraded form, resulting in a single band.
Experimental Conditions
Conditions during the IP experiment (e.g., washing, binding steps) might cause the loss of certain protein forms. For instance, proteins with weaker interactions may be washed away during the washing steps.
Antibody Specificity
The antibody used may bind exclusively to a specific form of the target protein, leading to the detection of only a single band in the IP sample.
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