Molecular biology of the cell - THEORY

Question 1

Explain how SDS-PAGE leads to separation of proteins according to their molecular weight. (5 marks)

Answer 1

• Proteins are separated by their molecular weight because they are treated to have the same charge-mass ratio. This is done by coating them in SDS which binds and denatures them and masks the charges of the R groups and gives the proteins a uniform negative charge. It also linearises the protein and breaks the hydrogen bonds.
• Then when a current is applied, the proteins migrate through the gel towards the positively charged electrode (anode).
• Smaller proteins will migrate faster
• The polyacrylamide gel is a dense mesh that means the larger the protein the slower its migration through the gel
• Coomassie blue dye then binds to the amino acid residues in acidic conditions to help visualise the proteins. The band intensity with Coomassie blue then correlates with the amount of protein present.
• However note they could be different proteins of a similar molecular weight