Molecular Diagnostics II: Protein Techniques

Question 1

Insulin has disulfide bonds

Answer 1

Post-translational modification, doesn’t happen in bacteria

Question 2

Antibodies recognize proteins

Answer 2

Antibodies made in response to antigens (antibody generators)

Question 3

Antibody structure

Answer 3

1. Heavy chain and light chain (Y-shaped)
2. Antigen binding site is at head of Y where heavy/light chains unite to form Fab domain

Fab = fragment antigen binding site
Chains linked by disulfide bonds

Question 4

Fc vs Fa

Answer 4

Fc = fragment that crystallizes, base of antibody
Fa = fragment with antigen binding site

Question 5

Epitope

Answer 5

Specific site of antigenic molecule that Fab region recognizes

Very specific

Question 6

Longer anti-body binding domain is more specific

Answer 6

10-11 AAs vs. 3-4 AAs

Question 7

Proteins are anti-genic

Answer 7

Can use rabbits to produce antibodies

Question 8

Multiple myeloma cell-line useful to make monoclonal antibody

Answer 8

Used to make large numbers of B-cells

Question 9

Preparation of monoclonal antibody

Answer 9

1. Inject antigen of interest in mouse. Remove its spleen.
2. Mix plasma cells from spleen with myeloma cells (hybridoma cells)
3. Isolate cells that produce desired antibody, develop pure cell line that produces a single antibody.

Question 10

Ascites fluid

Answer 10

1. Inject hybridoma cells into peritoneal cavity of mice

2. Tumor produces antibody-rich fluid called ascites fluid

Question 11

Monoclonal antibody purpose

Answer 11

1. For precise analytical and preparative reagents, used to purify proteins

Question 12

Polyclonal vs Monoclonal

Answer 12

Polyclonal = multiple antibodies expressed
Monoclonal = single antibody expressed

Question 13

Most sensitive amplification methods use an enzyme as a marker molecule attached to secondary antibody

Answer 13

Indirect detection has a stronger signal than direct detection

Question 14

Western Blot uses SDS-PAGE gel

Answer 14

NOT agarose, important to block membrane with neutral protein like casein or BSA

Question 15

ELISA (used for HIV test)

Answer 15

Enzyme linked immunosorbent assay

Presence of colored product indicates presence of antigen

Question 16

Sandwich ELISA

Answer 16

allows for detection and quantitation of antigen

Question 17

Indirect ELISA

Answer 17

HIV testing, wash unbound antibodies

Pregnancy test

Question 18

Immunofluorescence microscopy

Answer 18

Used to visualize subcellular localization of proteins

Stain antibody fluorescein (green) and rhodamine (red)

Question 19

Proteomics

Answer 19

Analysis of protein expression/alteration

Question 20

Liquid Chromatography

Answer 20

Filtering proteins by their charge and then filtering them by size. Large proteins come out first because small proteins get trapped in resin.

Question 21

Mass Spectrometry

Answer 21

Used to detect levels of protein in a sample AND unique modifications