10: Analytical Techniques

Question 1

what are analytical techniques

Answer 1

laboratory techniques to measure analytes of interest
predominantly use of molecular biology or analytical chemistry techniques
can be used on wide range of samples - plasma, serum, urine, faeces, tissue, cell lines

Question 2

1st molecular technique = plant based immunoassays

Answer 2

use antigen and antibody
commonly used to measure proteins
ELISA is used often - enzyme linked immunosorbent assay
used in research to quantitate inflammatory and stress responses

Question 3

sandwich ELISA

Answer 3

1. capture antibody added to sample plate
2. sample added to plate well
3. detection antibody added
4. conjugated antibody added
5. add substrate and measure assay response

Question 4

detection methods

Answer 4

colorimetry - wavelengths of light directed at plate and absorption measured (more colour = more absorbance)

fluorescence - fluorophore conjugated to antibody and specific wavelength or light directed at plate, fluorophore absorbs light and emits alternative wavelength (more analyte = more fluorophore = more light)

chemiluminescence - emission of light caused by chemical excitation of substrate (more analyte = more light)

Question 5

2nd molecular technique = western blotting

Answer 5

alternative immunology-based technique
measure proteins of interest
does not require uniquely specific antibodies for analyte due to gel separation
gel separation of proteins followed by immunostaining
semi-quantitative technique - can validate the presence of proteins
used often in research to assess muscle synthesis activation

Question 6

western blotting method

Answer 6

proteins denatured by boiling
put on well on an SDS-PAGE gel
separated by electrophoresis
transferred from gel to blotting membrane
antibody bound to protein and response is measured
(detection techniques same as ELISA)

Question 7

what are chemical techniques

Answer 7

focus on easurement of small molecules
small molecules measured are metabolites or peptides
methods developed based on chemical behaviour of target molecules
can measure multiple target metabolites in a single analysis

Question 8

chemical technique = chromatography

Answer 8

allows complex mixtures to be separated
reduces complexity of sample analysis
improves sensitivity by removing competing molecules from analysis
can separate isomeric molecules
gas or liquid chromatography used

Question 9

how chromatography works

Answer 9

separation achieved by passing sample through column
chemicals within column cause interactions with the molecules in sample = stationary phase
different molecules interact differently to different stationary phases
separated based on chemical affinity - how much the molecule wants to bind to stationary phase
suitability of stationary phase chosen based on interactions that occur between sample and phase
important that analyst chooses correct stationary phase, or the analysis may not be be accurate or reproducible

Question 10

measuring chromatography

Answer 10

interaction of analytes with stationary phase can be influenced by: temp, mobile phase flow rate, mobile phase composition
methods developed so analyte of interest exits column without interfering molecules
time of chromatography (retention time) is reproducible if conditions are repeated