Tools of Biochemistry Flashcards
Electrophoresis
separates molecules based on size, shape, and charge
pH and intrinsic props of molecules determine their charge
gel for support/sieve
molecules move from negative to positive pole
Native PAGE
proteins maintain stucture (non-denaturing)
migrate depending on size, shape, and native charge
example) Hb A migrates further than Hb S (less negative)
SDS-Page
denaturing
proteins treated: reducing agent to break disulfide bond, SDS denatures/binds to backbone and confers neg charge
separates protein by weight
larger size moves slower
ex) used to see if athletes are doping
Western Blotting
separates a protein
1) gel electrophoresis
2) transfer to paper (pos charge)
3) Label:
primary antibody to protein of interest (POI), secondary antibody with an enzyme attached, add substrate and enzyme will convert it to a colored or luminescent product where POI is located
Enzyme-Linked Immunosorbent Assay (ELISA)
differences compared to WB, purpose, steps, and indicator used
faster than a Western blot, sensitive to protein so use smaller amount
allows you to detect antibodies or antigens in a patient specimen
indicator: antibody with bound enzyme that can catalyze conversion of a colorless molecule to a colored one
direct or indirect (primary and secondary antibody)
the darker it is, the more it will flouresce or darker it will be
Enzyme-Linked Immunisorbent Assay (ELISA)
Steps:
1) apply the antibody or antigen depending on what you are testing
2) wash plate
3) add enzyme linked secondary antibody
4) at chromogenic substrate
HIV Diagnostic Testing Steps and Criteria for Interpretaton
If ELISA is POSITIVE: western blot and determine from there
if ELISA is NEGATIVE: negative test but if it is during the window period (time between infection and antibodies formed) you may have a false negative or indeterminant result (retest in 6 weeks)
criteria: two boxes, need to get one from each box
box 1- p31 or p24
box 2- gp160 or gp 120
ELISA and Heart Attacks
measures cardiac troponin T which is leaked from cells after injury
take serum from patients after MI and add antibody that can recognize cardiac troponin T
Co-Immunoprecipitation
shows proteins that interact
example) proteins A and B together, resolve via SDS page (now youe have A free and B free) western blot with an antibody against B
i. e. you target a known protein and pull out others in that complex and learn more about them
ex) figured out that MDM2 and p53 interacted (immunoprecip mdm-2 with anti mdm-2 serum and p53 proteins coprecipitated)
Immunoaffinity chromatography
purify proteins from cell extracts
pour solution in a column with an antibody that is attached to a bead
everything will wash through except what is bound
can be further separated via PAGE and IP
Immunohistochemistry
cell leaks, add a colored antibody to see what proteins are being expressed
(can do primary-secondary antibody with conjugated enzyme)
will produce a colored precipitate at the place in the tissue where the antibody/enzyme complex is located
ex) HER-2 immunistaining in breast cancer pt’s as a diagnostic tool
Epitope Tagging
add known epitope to a gene of interest
epitope- portion of a molecule to which an antibody binds and can be composed of sugars, lipids, or amino acids
epitopes are added to a molecule which an investigator wants to visualize
vizualize via gel, western blot, or label via immunofluorescence
can localize tagged protein within the cell
