Dear Researchers,
I am performing a Western Blot analysis. My protein's expected molecular weight was 96-122 kDa, but I observed it at 60-75 kDa.
Is there any problem with my protein preparation or something?
I need to know the reason for the fluctuation in molecular weight.
The molecular weight of a protein can vary from the expected molecular weight due to the presence of post-translational modifications (PTMs) such as phosphorylation, glycosylation, and other modifications that can alter the structure and properties of the protein. Additionally, the presence of proteases can cleave the protein into smaller fragments, changing its molecular weight. Variations in the pH of the environment may also influence the charge of the protein, causing the molecular weight to differ from the expected value.
Hello Premkumar Natraj
There are many reasons that could account for differences between observed and the expected protein molecular weight in gel electrophoresis and Western Blotting.
1. Whenever disruption of cell membranes is carried out during tissue homogenization or cell lysis there is a release of proteases that can degrade protein of interest. Proteins can undergo non-specific proteolytic digestion if one does not handle the sample correctly. Proteases that are released during cell lysis or tissue extraction (if protease inhibitors are not added or if cell lysis is not performed under cold condition) can cause protein fragmentation, resulting in smaller fragments of lower molecular weights.
2. Shifts can occur due to intrinsic net charge. In SDS-PAGE the intrinsic charge of proteins is essentially negligible when compared to the negative charge of SDS. When constant electric field is applied, proteins (which are coated with the negatively-charged SDS) migrate towards the anode. However, in certain cases, there is a possibility that a protein may contain a large number of positively charged arginine or lysine residues, which could affect SDS binding and influence migration of the protein in gel electrophoresis.
3. 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. In these cases, you will find that the observed molecular weight will be substantially higher than the predicted monomeric weight.
4. 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, or you may get proteins of lower molecular weight owing to premature stop codons.
5. Many proteins that are synthesized with a signal peptide, pro-peptide, or both are subsequently cleaved by specific proteases to generate mature protein which will result in a low molecular weight protein.
6. Post-translational modifications is another factor that may increase the molecular weight of the protein of interest.
You need to find the exact cause by considering these above factors which could help you solve your problem.
Best.
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