Tools of Protein Biochemistry

Question 1

what do you predict would move farther towards the positive electrode in gel electrophoresis, HbS (sickle cell hemoglobin) or HbA (adult WT hemoglobin)?

Answer 1

HbA would move faster/farther - glutamic acid is negatively charged

HbS has E->V mutation, and valine is hydrophobic (uncharged)

[electrophoresis only removes charge when SDS-PAGE is used]

Question 2

what are proteins treated with in denaturing gel electrophoresis?

Answer 2

1. reducing agent - breaks disulfide bonds
2. SDS (sodium dodecylsulfate) - anionic detergent - denatures protein and binds protein backbone to confer a uniform negative charge

Question 3

HbA is 65kD. What band(s) would you expect to see after running it through an SDS PAGE gel electrophoresis?

Answer 3

hemoglobin is tetramer of 2 alpha, 2 beta subunits

ionic bonds hold subunits together, so these would be denatured by SDS

you would see 4 bonds at ~15kD (so one thick band)

Question 4

what is the basic principle of western blotting

Answer 4

primary antibody that already exists in an animal against the variable region (Fab) of Ig

secondary antibody (for visualization) against constant region of antibody of the animal used for primary antibody

SDS gel electrophoresis —> transfer proteins to membrane —> block non-specific sites —> primary antibody —> secondary antibody —> imaging

Question 5

what can ELISA be used to detect?

Answer 5

enzyme-linked immunosorbent assay (ELISA): can detect antibodies or antigens

coat onto microplate, add 1st/2nd antibody, read absorbance spectrophotometer

Question 6

you see a patient in your clinic that presents with symptoms and history consistent with an HIV infection. You order an ELISA and it comes back positive. What do you tell the patient?

Answer 6

ELISA is very sensitive, so therefore not as specific (may be cross-reacting antibodies)

need to confirm with Western blot, which is more specific (but more labor intensive)

Question 7

what is the difference between what you put in ELISA vs what you put in a Western blot?

Answer 7

ELISA - put entire protein mixture into microplate, but can’t differentiate what is binding what

Western - separate proteins, so more specific information (also more labor intensive)

Question 8

where will the target protein be found following immunoprecipitation?

Answer 8

in the PRECIPITATE (don’t overthink it)

junk is in the supernatant

Question 9

how does co-immunoprecipitation (Co-IP) work? (concept)

Answer 9

when proteins are interacting, the precipitate will pull down both

used to identify protein-protein interactions

Question 10

explain (simply) immunohistochemistry

Answer 10

antibodies detect target protein in situ sample (fixed tissue)

secondary antibody conjugated to enzyme (ex: HRP, horseradish peroxidase) or fluorescent tag

Question 11

in Western blotting to detect HIV:
gel electrophoresis and blot is performed on ____,
blot is incubated with _____,
and blot detects _____

Answer 11

performed on HIV protein standards
blot incubated with patient serum
blot detects presence of anti-HIV antibodies in serum