Analytical Techniques

Question 1

State an example of a plate based immunoassay.

Answer 1

Enzyme Linked Immunosorbent Assay (ELISA)

Question 2

Put these in order from first to last in the ELISA process:

Blocking solution, detection antibody, capture antibody, substrate solution, QC/sample, stop solution

Answer 2

1. Capture antibody
2. Blocking solution
3. QC/sample
4. Detection antibody
5. Substrate solution
6. Stop solution

Question 3

What is the first step of an ELISA assay?

Answer 3

Capture antibody is attached to sample plate - antibody specific and binds with chosen analyte.

Question 4

After the sample is added to the well, why is liquid removed and the well washed?

Answer 4

Washes away any unbound samples.

Question 5

By washing away unbound samples in an ELISA assay, what increases?

Answer 5

Specificity of the experiment.

Question 6

What two antibodies create a sandwich around the sample, in step 3 of an ELISA assay?

Answer 6

Capture and detection antibody.

Question 7

If an antibody is described as conjugated, what does this mean?

Answer 7

Something is attached to the antibody, such as an enzyme.

Question 8

How is the assay response measured in an ELISA experiment?

Answer 8

Addition of a substrate/chemical that will give a known response in presence of the conjugated antibody.

Question 9

When a well turns a deeper colour in a well, how much light reaches the detector, compared to that of a lighter well?

Answer 9

Less light reaches the detector as more light is absorbed in the sample.

Question 10

If a well in an ELISA experiment has a higher absorbance level, what does this mean for protein?

Answer 10

Higher absorbance level = higher concentration of protein.

Question 11

What compound absorbs light and emits and an alternative wavelength?

Answer 11

Fluorophore.

Question 12

Why is the use of a fluorescence carried out in a black well?

Answer 12

Some light from the fluoresce will be lost to the wells in a clear plate (may cause cross contamination/faulty recordings).

Question 13

What is chemiluminescence?

Answer 13

The emission of light caused by chemical excitation of substrate.

Question 14

Western blotting is used to what?

Answer 14

Measure proteins of interest in tissue samples.

Question 15

Which analytical technique does not require the use of antibodies?

Answer 15

Western blotting.

Question 16

Western blotting is a semi quantitative technique, what does this mean?

Answer 16

It only validates the presence of a protein.

Question 17

Denatured sampled are loaded into a well with what gel for electrophoresis?

Answer 17

Polyacrylamide gel

Question 18

The porous cross linked structure of the electrophoresis gel allows what?

Answer 18

Proteins to pass through.

Question 19

The gel for electrophoresis is surrounded by a conductive buffer allows for what?

Answer 19

The movement of electrons.

Question 20

Which proteins travel faster and further up the electrophoresis plate?

Answer 20

Small

Question 21

Why are proteins transferred from the gel to blotting membrane?

Answer 21

Visualisation.

Question 22

Describe the process of immunostaining.

Answer 22

1. Primary antibody binds to epitope (given region) on protein of interest.
2. Secondary antibody added and binds to a specific site on primary antibody.
3. Secondary antibody conjugated to reported molecule.
4. Detected by same techniques as ELISA.