Analytical Techniques Flashcards
(22 cards)
State an example of a plate based immunoassay.
Enzyme Linked Immunosorbent Assay (ELISA)
Put these in order from first to last in the ELISA process:
Blocking solution, detection antibody, capture antibody, substrate solution, QC/sample, stop solution
- Capture antibody
- Blocking solution
- QC/sample
- Detection antibody
- Substrate solution
- Stop solution
What is the first step of an ELISA assay?
Capture antibody is attached to sample plate - antibody specific and binds with chosen analyte.
After the sample is added to the well, why is liquid removed and the well washed?
Washes away any unbound samples.
By washing away unbound samples in an ELISA assay, what increases?
Specificity of the experiment.
What two antibodies create a sandwich around the sample, in step 3 of an ELISA assay?
Capture and detection antibody.
If an antibody is described as conjugated, what does this mean?
Something is attached to the antibody, such as an enzyme.
How is the assay response measured in an ELISA experiment?
Addition of a substrate/chemical that will give a known response in presence of the conjugated antibody.
When a well turns a deeper colour in a well, how much light reaches the detector, compared to that of a lighter well?
Less light reaches the detector as more light is absorbed in the sample.
If a well in an ELISA experiment has a higher absorbance level, what does this mean for protein?
Higher absorbance level = higher concentration of protein.
What compound absorbs light and emits and an alternative wavelength?
Fluorophore.
Why is the use of a fluorescence carried out in a black well?
Some light from the fluoresce will be lost to the wells in a clear plate (may cause cross contamination/faulty recordings).
What is chemiluminescence?
The emission of light caused by chemical excitation of substrate.
Western blotting is used to what?
Measure proteins of interest in tissue samples.
Which analytical technique does not require the use of antibodies?
Western blotting.
Western blotting is a semi quantitative technique, what does this mean?
It only validates the presence of a protein.
Denatured sampled are loaded into a well with what gel for electrophoresis?
Polyacrylamide gel
The porous cross linked structure of the electrophoresis gel allows what?
Proteins to pass through.
The gel for electrophoresis is surrounded by a conductive buffer allows for what?
The movement of electrons.
Which proteins travel faster and further up the electrophoresis plate?
Small
Why are proteins transferred from the gel to blotting membrane?
Visualisation.
Describe the process of immunostaining.
- Primary antibody binds to epitope (given region) on protein of interest.
- Secondary antibody added and binds to a specific site on primary antibody.
- Secondary antibody conjugated to reported molecule.
- Detected by same techniques as ELISA.
