in vitro experimentation; proteins, DNA-proteins interactions Flashcards
how can we separate proteins?
using SDS-PAGE
how can we detect proteins?
using western blot, immunofluorescence, immunohistochemistry, ELISA and FACS
how can we study protein complex interactions?
by using co-immunoprecipitation
how can we study DNA-protein interactions?
by using luciferase assats, EMSA and ChIP
priniciple of SDS-PAGE
separation of proteins based on the molecular weight
principle of Western Blot, ELISA, IHC, IF and FACS
detection of proteins using specific antibodies
what is SDS-PAGE?
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
general process of SDS-PAGE
- proteins are negatively charged (due to SDS) and move to positive electrode
- proteins separate by size (molecular weight)
- smaller proteins move faster
what is needed to carry out an SDS-PAGE?
- acrylamide gel
- protein sample
- loading dye (density + dye)
- SDS-based solution
how can we identify the proteins when we remove the gel from the tank?
using a dye of Coomassie Blue Stained
using antibodies to detect proteins
- antibodies vary in specificity but ideally they only recognise one epitope
applications for antibodies
basic research, diagnostics, therapeutics
process of western blotting
- isolate all proteins form sample
- gel electrophoresis
- transfer to membrane
- block
- primary antibody
- secondary antibody
three ways of protein detection in western blot
- colorimetric
- chemiluminescence
- fluorescence
what is the ELISA assay?
enzyme-linked immunabsorbent assay