BIOL3612 Lab 5A: Separation of Proteins by SDS-PAGE & Concentration Determination via Spectrophotometry

Why was A280 used in this experiment?

Quick way of obtaining an indication of protein concentration in a sample compared to the longer Lowry method used previously

Cysteine side chains

Cysteine’s side chain ends in a sulfhydryl (-SH) group

Oxidized form of sulfhydryl

Some proteins contain disulfide bridges that covalently link 2 cysteine residues to each other (S-S)

• Electrons are lost during the formation of the disulfide bridge

How to break disulfide bonds

Experimentally, these bonds can be broken (reduced again) in the presence of an excess of a reducing agent that has its own sulfhydryl groups

• EX.: beta-mercaptoethanol is a popular reducing reagent for this purpose 

Why is breaking disulfide bonds desirable?

• Breaking disulfide bonds is desirable in typical “reducing” SDS-PAGE, when the purpose is to separate polypeptide chains by size

  • Removing any disulfide bridges that hold 2 chains together or that hold regions of 1 individual chain together 

What happens if disulfide bonds are not removed in SDS-PAGE?

• It would be possible to perform nonreducing SDS-PAGE, in which the disulfide bonds were kept intact, however, migration would no longer be purely based on polypeptide chain length 

What does PAGE stand for in SDS-PAGE?

PAGE stands for polyacrylamide gel electrophoresis

• The gel is composed of polyacrylamide, a polymer of acrylamide 

• When a current is applied, small proteins (now coated with negative charge) can move more easily through the polymer towards the positively-charged anode, while larger proteins are more impeded in their migration through the gel matrix and travel slowly 

Sodium dodecyl sulfate (SDS)

• Anionic detergent with a sulfate end (the anion) and a hydrocarbon tail  

  • Serves as a denaturant, disrupting NONcovalent interactions within the protein (van der Waals, hydrophobic, H-bond, electrostatic) 

  • Also serves to evenly coat the unfolded proteins with negative charge, masking the inherent charges from the R-groups and instead providing a negative charge that is proportional to their length 

What does loading buffer contain?

1. SDS

2. A reducing agent such as beta-mercaptoethanol

3. A dye to help track the migration of the sample

4. A dense substance such as glycerol to help the sample sink in to the wells as it is loaded