There are several possible reasons for this, including: (1) incorrectly diluting the protein sample, (2) inadequate transfer of proteins onto the blotting membrane, (3) protein degradation, (4) incorrect antibody concentrations, (5) improper blocking of the membrane, (6) nonspecific binding of the antibody, (7) presence of interfering substances in the sample, and (8) cross-reactivity of the antibody with other proteins in the sample. It is important to review the experimental conditions and troubleshoot the issue to identify the cause.

Proteins can appear at different sizes due, for example, specific amino acid composition, post translational modifications, poor denaturing and others. Really depends on your protein. If you have a positive control and it runs at the same size then there's nothing to worry about.

Hello Khawla Saif

You could consider the following reasons for the protein bands to be higher in size than expected.

1. In denaturing SDS-PAGE, most protein complexes disassociate during sample preparation and electrophoresis, and component proteins run as monomers. However, in certain cases, some complexes may not be completely disrupted, and some proteins may still remain in their homo- or heteromeric complexes even after subjecting the protein sample to SDS and β-mercapto ethanol treatment. Therefore, dimers, multimers, or protein-protein interactions may occur because samples have not been fully reduced or denatured. In these cases, you will find that the observed molecular weight will be substantially higher than the predicted monomeric weight.

2. Splice variants, one gene can generate proteins of different sizes due to alternative splicing during mRNA maturation. This can result in additional protein-coding sequences and as a result higher molecular weight protein products may be observed.

3. Post-translational modifications is another factor that may increase the molecular weight of the protein of interest. The common examples are glycosylation in which several proteins can undergo glycosylation such as N-linked glycosylation and O-linked glycosylation, where sugar moieties are covalently attached to the polypeptide chain. This results in a molecular weight that is higher than one calculated based on protein sequence. Phosphorylation which involves the addition of a single phosphoryl group contributes only slightly to the molecular weight, but if phosphorylation occurs at multiple sites then it can lead to more noticeable molecular weight changes. Ubiquitination which involves binding of ubiquitin to protein may occur as mono-ubiquitinated or poly-ubiquitinated. These may result in molecular weight changes.

4. Sometimes, a protein may have large number of arginine or lysine residues and as a result it may remain positively-charged. This could affect SDS binding and influence migration of the protein in SDS-PAGE gel, creating a discrepancy between observed and calculated molecular weight.

Best.