1. It's usually difficult to identify a protein of interest in a Commassie Blue-stained gel of cell extracts Coomassie Blue-stained gel MW stds. Cell extracts 33 44 78 18 132 Size (kD) Western blots take advantage of the sensitivity of antibodies to visualize proteins of interest in complex preparations

2. How are epitope tags used to locate proteins on western blots? What steps are involved in western blots?

3. Proteins are expressed from plasmids that encode epitopes in the same reading frame with the cloned sequence Protein of interest Epitope added by plasmid sequences Antibody that binds the epitope Epitopes add antibody binding sites to proteins

4. 9 AA ORF His 6 HA antibody binding domain of S. aureus protein A 14 AA ORF His 6 V5 pBG1805-encoded proteins have multiple epitopes, which add 19 kDa to the protein’s MW pYES2.1-encoded proteins have two epitopes, which add ~5 kDa to the protein’s MW

5. Epitope-tagged proteins on gel 1. Primary antibody binds proteins (stoichiometric) 2. Secondary antibodies recognize multiple sites on primary antibody 3. Enzyme or fluorochrome amplifies the signal Western blots typically use multiple antibodies to visualize epitope- tagged proteins

6. HRP Horseradish peroxidase (HRP) 40,000 MW enzyme Catalyzes reaction that produces a colored product Blue reaction product 3,3'5,5'-tetramethyl benzidine + H 2 O 2 HRP-catalyzed reaction amplifies the signal and generates a visible product

7. 14 AA ORF His 6 V5 pYES2.1 9 AA ORF His 6 HA IgG binding domain pBG1805 Problem: How can proteins encoded by pBG1805 and pYES2.1 plasmids be visualized on the same gel? Sensitive (and expensive) monoclonal antibodies are available for the viral HA and V5 epitopes The His 6 tag is useful for purifying proteins, but it is a poor antigen

8. Staphylococcus aureus Protein A A solution: Use an alternative strategy to identify proteins encoded by pBG1805, based on the IgG binding domain of S. aureus protein A Protein A is a transmembrane protein with multiple binding sites for the F c portion of IgGs Helps bacterium to evade the host’s immune system during infections

9. Different strategies will be used to detect pBG1805- and pYES2.1- encoded proteins pYES2.1 -encoded proteins on gel Mouse monoclonal Ab detects V5 antigen HRP pBG1805-encoded proteins on gel HRP HRP-conjugated rabbit anti-mouse IgG polyclonal antibody

10. How are epitope tags used to locate proteins on western blots? What steps are involved in western blots?

11. A multi-layer "sandwich" of filter papers and sponges encloses the gel and the PVDF membrane within a transfer cassette

12. The cassette with its "sandwich" is inserted into the transfer apparatus Be mindful of the correct polarity! Black plate of sandwich toward black side of cassette holder The electric current transfers proteins from the gel to the membrane

13. SDS-PAGE gel PVDF membranes are hydrophobic. To bind proteins, they must first be wetted with methanol, followed by water and transfer buffer Proteins are electrophoretically transferred to a PVDF (essentially Teflon ) membrane

14. Blocking step PVDF membranes will bind proteins nonspecifically. Milk contains high concentrations of casein proteins, which bind and saturate these low affinity, nonspecific sites on the membranes. Epitope-tagged proteins (invisible) MW standards Membranes are incubated with 5% nonfat milk MW standards

15. Primary antibody binding step Primary antibody is a mouse monoclonal antibody that recognizes the V5 epitope 1 2 3 1 – MW standards 2 – proteins with V5 epitope tag (pYES2.1) 3 – proteins with protein A domain (pBG1805) Antibodies bind with high affinity to V5 epitopes

16. 1 – MW standards 2 – proteins with V5 epitope tag (pYES2.1) 3 – proteins with protein A domain (pBG1805) Secondary antibody is a rabbit polyclonal antibody that: recognizes the constant region of the mouse IgGs (lane 2) binds to S. aureus Protein A (lane 3) 1 2 3

17. 1 – MW standards 2 – proteins with V5 epitope tag (pYES2.1) 3 – proteins with protein A domain (pBG1805) Detection Blot is incubated with substrates for HRP 1 2 3 Blot is incubated until reaction products appear

18. Blue-black bands indicate the position of epitope-tagged proteins Western blot done by BC students

19. Process begins with the electrophoretic transfer of proteins to a membrane Blockin g Membrane replica Primary antibody Secondary antibody Enzymatic detectionFinal blot