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
chemical techniques
measure small molecules
metabolites / peptides
methods based on chemical behaviour of target molecules
capable of measuring multiple metabolites
basics of chromatography
chemical properties to separate complex mixtures
reduces complexity of sample analysis
can separate isomeric molecules
why does chromatography improve sensitivity
removes competing molecules
2 types of chromatography
gas or liquid (GC LC)
chromatography process
sample passes through column
chemicals in column cause interactions with molecules
different molecules react differently (based on chemical affinity - how much they want to bind)
what are the chemicals called in the column in chromatography
stationary phase
how are stationary poses chosen
based on interactions that occur between sample and the phase
why must the correct stationary phase be chosen
to make sure its accurate and reproducible
what influences interactions in chromatography
temp
mobile phase flow rate
mobile phase composition (pH - LC only)
what influences interactions in chromatography
temp
mobile phase flow rate
mobile phase composition (pH - LC only)
