Protein Analysis Techniques

Question 1

methods for analyzing protein production

Answer 1

1. activity assays
2. PAGE/SDS-PAGE
3. western blot
4. ELISA
5. immunohistochemistry
6. LC-MS/Proteomics

Question 2

activity assays

Answer 2

doing simple reaction assay (analyzing substance) that is attempting to determine how active an enzyme is
- enzyme and substrate of enzyme mixed together –> then measure end product

Question 3

PAGE/SDS-PAGE

Answer 3

denatured or non-denatured (native) protein extracts separated by PAGE
- polyacrylamide gel electrophoresis/sodium dodecyl-sulfate polyacrylamide gel electrophoresis –> difference: SDS denatures protein so they all have same charge –> proteins can have different charges –> but can’t work with denatured proteins after SDS-PAGE
- just gel electrophoresis for proteins
- denatured protein separated by PAGE
- add dye after gel has been run –> Coomassie blue or silver stain

Question 4

western blotting

Answer 4

for protein detection and semi-quantitative
1. denatured or non-denatured protein extracts separated by PAGE
2. separated proteins transferred to membrane
3. fluorescently labeled (or enzyme-linked) antibodies (probes) added to membrane and bind to target proteins
- for enzyme-linked antibodies, chemiluminescent substrates added
5. signal detected by autoradiography

Question 5

immunoassays + define antibody, antigen, and epitope**

Answer 5

protein detection; biochemical assays (tests) that measure presence of specific proteins using antibodies as probes
- examples: western blotting, ELISA, and immunohistochemistry
- antibody = probe; large, Y-shaped protein that is produced by immune cells in response to foreign substance to tag and neutralize a foreign substance
- antigen: foreign target of an antibody
- epitope: specific area of antigen where antibody binds

Question 6

types of immunoassays

Answer 6

1. Direct assay: uses only 1 antibody probe
   - antibody binds directly to target compound
2. Indirect assay: uses 2 antibodies
   - 1st/primary antibody binds to antigen –> secondary antibody binds to primary antibody –> trying to avoid false positive

in both cases, antibodies have fluorescent or other visual indicator

Question 7

ELISA

Answer 7

enzyme-linked immunosorbent assay
- uses immunoassays
- quantitative unlike westerns and dot blot
- direct and indirect tests
steps: antigens at the bottom of a plate well –> add specific primary antibody that binds to antigen –> then enzyme-linked secondary antibodies bind to primary antibody –> add substrate to well which enzyme will process and produce colored signal
– wash between each step to remove any unwanted antibodies

Question 8

immunohistochemistry (IHC)

Answer 8

microscopic = produces digital image; technique that employs antibodies to examine in situ location of a specific protein(s) in cells
- basically immunoassay process (with both direct and indirect) but looking at cell tissue

Question 9

genomics vs. transcriptomics vs. proteomics

Answer 9

genomics: study of organism’s genome - complete set of DNA
transcriptomics: study of transcriptome - complete set of RNA molecules
proteomics: study of proteome - entire set of proteins

Question 10

LC-MS

Answer 10

liquid chromatography/mass spectrometry
- protein sequencing method

Question 11

southern blotting vs. northern blotting vs western blotting

Answer 11

southern: separated DNA, nucleic acid hybridization
northern: separates RNA, nucleic acid hybridization
western: separates proteins, antibody probe/immunoassay