Antibody Based Experimental Techniques

Question 1

Explain western blot and what it identifies

Answer 1

IDs protein or set of proteins within a mixture

1. SDS-PAGE
2. Filter paper exposed to antibody to bind to protein
3. Wash excess
4. 2nd Ab added (attached to enzyme) specific for Fc region of primary antibody, leads to visible colored antibody

Question 2

Explain ELISA

Answer 2

= enzyme-linked immunosorbent assay
Quantitative, detects Ag or Ab and is used to diagnose diseases
Microtiter plates with protein sample, then like western blot and spectrophotometer to measure color

Question 3

Explain immunoaffinity chromatography

Answer 3

Purification of macromolecules

Ab in chromatography, captures Ag, Ag eluted by changing pH of salt washing buffer

Question 4

Explain innumofluorescence microscopy

Answer 4

A fluorescent labeled Ab to detect distribution and structure of cellular components

Question 5

Explain fluorescence activated cell sorting (FACS)

Answer 5

Semi-quantitative, analysis of Ag expression in cellular populations
Labeled cells are physically separated and counted
Can ID leukocyte types based on CD molecule expression