What are the common causes of an unexpected size of western blot band?

Unexpectedly low molecular weight (MW) bands in protein analysis can arise from various sources including splice variants, nontarget proteins with similar epitopes, and protein cleavage or degradation. Proteins may undergo cleavage by proteases or degradation processes, resulting in the generation of smaller fragments with lower molecular weights than the intact protein. Alternative splicing of mRNA can generate protein isoforms with different structural features, including variations in molecular weight. Cross-reactivity with nontarget proteins that share similar epitopes with the target protein can result in the appearance of bands at lower molecular weights than expected.

Unexpectedly high molecular weight bands in protein analysis can arise from various factors. One factor is due to incomplete reduction or denaturation of samples, which can lead to the formation of multimers, dimers, or protein-protein interactions. Additionally, if a protein appears at a slightly higher molecular weight, it could indicate that the protein has undergone post-translational modifications at one or more amino acid residues.

Several factors can contribute to the presence of multiple nonspecific bands on a blot. These factors include inadequate blocking, excessive antibody concentration, poor quality antibodies, or nonspecific binding caused by the presence of SDS. When antibody concentration is too high, there is an increased likelihood of binding to unintended targets. Antibodies of lower quality may lack specificity, leading to nonspecific binding with multiple proteins present in the sample. Incomplete or insufficient blocking of nonspecific binding sites on the membrane can result in the binding of antibodies to non-target proteins, leading to the generation of nonspecific bands. Residual SDS in the sample or buffers may contribute to nonspecific binding of antibodies, leading to the appearance of multiple bands on the blot.