Question

  You work with a group of scientists aiming to understand a specific ER protein that has a molecular weight of 75kDa. You ask your intern to mix the ribosomes and mRNA together, purify the resulting protein, and run electrophoresis and a western blot. After completing the western blot experiment, your intern is worried because they did everything you told them to, but continues to find a protein at a higher molecular weight. The intern finally gathers the courage to approach you to tell you about the failed experiment. What do you tell your intern? Why did the protein appear at a higher molecular weight? What experiment should you have done?

Answer 1

The most probable reason behind getting higher molecular weight of protein as compared to the actual one (75Kda) is due to modification of proteins after translation (attachment of function chemical group to the polypeptide. The posttranslational modifications like glycosylation (attachment of glycan) which increase the size of protein significantly and when we characterize then get high molecular weight of protein. This post translation modification is essential for the proper folding of the proteins so that they can act normally in the cell and these proteins run slower on the gel as compared to the non-glycosylated one.  To rule of such kind of observation, you should have treated your protein samples with chemical or enzyme, which can help in Deglycosylation, and it is recommended to use before loading samples on SDS-PAGE.