Analytical techniques Flashcards
2 molecular techniques
plate based immunoassays
western blotting
what’s involved in plate based immunoassays
antigen antibody
applied to measuring proteins
elisa
Elisa method
- capture antibody attached to sample plate and use bovine serum to block non specific sites
- add sample to plate and wash so only analyte of choice can bind
- detect antibody added
- conjugated antibody added to detect antibody
- add substrate and measure change of colour
detection methods
colorimetry
fluorescence
chemiluminescence (emission of light caused by chemical excitation)
when are immunoassays used in exercise research
to quantitate inflammatory and stress responses
basics of western blotting
doesn’t require uniquely specific antibodies for analyte
gel separation of proteins then immunostaining
how and why do you denature proteins first in western blotting
boiling sample to separate based solely on molecular size
what do you do after denaturing proteins in western blotting
load into well in a polyacrylamide gel and undergo electrophoresis
most common format of electrophoresis
SDS-PAGE
SDS-PAGE separation process
gel is porous cross linked = proteins pass through
SDS used to give protein backbones negative charge
gel surrounded by conductive buffer
current passes across
-ve proteins travel down
smaller move quicker = further
where do proteins move towards in SDS-PAGE separation
to the anode
blotting process
proteins move to blotting nitrocellulose membrane
charge used to direct proteins put gel onto membrane
non specific binding sites blocked by bovine serum
staining
immunostaining process
primary antibody binds to epitope (region) of interest
secondary antibody added binding to site on primary antibody
secondary antibody conjugated to reporter molecule
detected some way as Elisa
why do primary antibodies not need to be highly specific in western blotting immunostaining
gel separation has been performed
what samples does western blotting typically use
tissue
assessing muscle synthesis activation
