Molecular Diagnostics II: Protein Techniques Flashcards
Insulin has disulfide bonds
Post-translational modification, doesn’t happen in bacteria
Antibodies recognize proteins
Antibodies made in response to antigens (antibody generators)
Antibody structure
- Heavy chain and light chain (Y-shaped)
- 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
Fc vs Fa
Fc = fragment that crystallizes, base of antibody Fa = fragment with antigen binding site
Epitope
Specific site of antigenic molecule that Fab region recognizes
Very specific
Longer anti-body binding domain is more specific
10-11 AAs vs. 3-4 AAs
Proteins are anti-genic
Can use rabbits to produce antibodies
Multiple myeloma cell-line useful to make monoclonal antibody
Used to make large numbers of B-cells
Preparation of monoclonal antibody
- Inject antigen of interest in mouse. Remove its spleen.
- Mix plasma cells from spleen with myeloma cells (hybridoma cells)
- Isolate cells that produce desired antibody, develop pure cell line that produces a single antibody.
Ascites fluid
- Inject hybridoma cells into peritoneal cavity of mice
2. Tumor produces antibody-rich fluid called ascites fluid
Monoclonal antibody purpose
- For precise analytical and preparative reagents, used to purify proteins
Polyclonal vs Monoclonal
Polyclonal = multiple antibodies expressed Monoclonal = single antibody expressed
Most sensitive amplification methods use an enzyme as a marker molecule attached to secondary antibody
Indirect detection has a stronger signal than direct detection
Western Blot uses SDS-PAGE gel
NOT agarose, important to block membrane with neutral protein like casein or BSA
ELISA (used for HIV test)
Enzyme linked immunosorbent assay
Presence of colored product indicates presence of antigen
Sandwich ELISA
allows for detection and quantitation of antigen
Indirect ELISA
HIV testing, wash unbound antibodies
Pregnancy test
Immunofluorescence microscopy
Used to visualize subcellular localization of proteins
Stain antibody fluorescein (green) and rhodamine (red)
Proteomics
Analysis of protein expression/alteration
Liquid Chromatography
Filtering proteins by their charge and then filtering them by size. Large proteins come out first because small proteins get trapped in resin.
Mass Spectrometry
Used to detect levels of protein in a sample AND unique modifications
