Pre-lab Immunology practical

Question 1

What is SDS Page and what is it used for?

Answer 1

-Sodium Dodecyl Sulfate-Polyacrylamide gel Electrophoresis (SDS-PAGE) is a technique used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their molecular weigh

-SDS-PAGE only separates the proteins on a gel matrix, but these are still invisible

• It is a method of protein analysis

Question 2

How does SDS-PAGE work?

Answer 2

1. Mix proteins with SDS Which breaks all the non-covalent bonds so it disturbs all the hydrogen, ionic interaction and hydrophobic interactions in all proteins
2. It binds in a specific way to proteins depending on the mass of the protein
3. Denatures proteins and make them linear

4.Add a negative charge because the SDS has a negative charge

5. The protein goes from 3d shape to a linear shape with all negative charges
6. 2 variable are removed from the protein mixture- the variable that different proteins will have different shape (Now linear all the same) 2 Variable proteins will have different charges(Mixture with SDS (now they all have the same charge which is negative)
7. Now it is easier and more effective to separate the protein in the electrical field
8. They separate in the electric field by their size - large/longer linear proteins ad have a high molecular weight - short/smaller linear proteins are closer to the anode side (+) and have a low molecular weight

Question 3

What is Coomassie Brilliant Blue Staining?

Answer 3

-Technique that uses a dye that aspecifically binds proteins, to detect them on a gel

-Quick and useful to assess the gel, before performing additional tests(i.e. Western blot)

-Proteins appear as bands, and the intensity of the band depends on the abundance and size

Question 4

What is the Coomaasie staining process?

Answer 4

-Add stain and was with water to see the proteins
-MW helps determine the measurements of the other bands in other samples

Question 5

What is Western Blotting and its use?

Answer 5

-Western blotting is used to visualise and identify proteins after these have been separated on a gel based on their differential size

-The protein samples are transferred (electrophoretically) from a gel to a membrane and are detected via a specific antibody

-It is used to find the viral spike protein among thousand of proteins

Question 6

What is the process of the Western blot?

Answer 6

1. Transfer the protein samples from the gel to a membrane (like a special white paper)

2.Use the electric field to transfer to the paper membrane - Membrane has the copy of what was on the gel

3. Staining- primary antibody is directed against the viral spike protein (It has be made to only bind to the viral spike protein)

4.Mix the membrane with the primary antibody and if the viral spike protein is present the primary antibody will bind to it only

5.Secondary antibody bind to primary antibody and has an enzyme, the enzyme is proroxase oxidases the substrate and a colour can be made

6.If the western blotting works you can see in the visualisation - the dark bands means the detection of the specific protein among thousand of proteins on the gel

Question 7

How can you tell the largest MW and smallest MW?

Answer 7

Largest MW - Moved the least
Smallest MW- Moved the most

Question 8

What is the principle of SDS-PAGE?

Answer 8

-SDS-PAGE is a detergent that denatures proteins through binding to hydrophobic regions
-Small proteins migrate faster through the gel under the influence of electric field and larger proteins take longer due to sieving effect of the gels

Question 9

What are the principles of Coomassie Staining?

Answer 9

It contains -Glacial acetic acid (enhances Coomassie specific binding), Methanol for denature proteins so they do not ’escape from the gel’

Destaining - use of water

Question 10

What are the principles of the Western Blot?

Answer 10

-Order of it -1.Cathode,2. reservoir stack,3. gel,4.Blotting membrane, 5. bottom reservoir stack&6. Anode

-Filter paper (wet with transfer buffer) is added on top of the gel and under the membrane, to allow transfer

-Secondary antibody - makes light substrate which is what makes dark bands on the western blot (Captured on camera)

Question 11

In SDS-PAGE, which of the following is a primary factor that dictates how far a protein will migrate?

Answer 11

Size

Question 12

You ran three samples for SDS-PAGE and thenperformed Comassie staining, what is the most likelyconclusion you can draw?

Answer 12

C) Protein in Lane C could be a monomer or a proteinwith subunits of the same size

Question 13

Western blotting is used to detect?

Answer 13

Specific protein in a sample

Question 14

1)What is ELISA?
2)What does it do?

Answer 14

1) Enzyme-Linked ImmunoSorbent Assay

2) Very sensitive immunochemical technique which is used to access the presence of specific protein (antigen or antibody) in the given sample and quantification

Question 15

What is the difference between specificity and affinity?

Answer 15

*Specificity is the ability to discriminate among diverse proteins.

• Affinity is the ability to tightly bind to molecules.

Question 16

What is the the Indirect ELISA Method?

Answer 16

1.Binding Known Antigen - a sample of known antigen being bound to the wells

2. Blocking - The other unoccupied sites in each well are then bound by a concentrated solution of non-interacting protein,
3. Washing – Rinse to remove any unbound antigen and non-interacting protein.
4. Adding Test Sample Primary Antibody - The test sample of serum containing the primary antibodies is added to each well. Antibodies could be HIV,
5. Washing – Rinse to remove any antibodies that did not bind to the known antigen.

Question 17

What is part 2 of indirect ELISA method?

Answer 17

6. Adding Enzyme-linked Secondary Antibody - An enzyme-linked secondary antibody is added next to bind to the test sample antibodies. The enzyme on the secondary antibodies are proteins, such as horse radish peroxidase or alkaline phosphatase.
7. Washing – Rinse to remove any secondary antibodies that did not bind to the primary antibody
8. Adding Substrate - A substrate is then applied which is converted by the enzyme to give a color or fluorescence or electrochemical signal. the presence of horse radish peroxidase= green, OPD= orange, and TMB =blue. In the presence of alkaline phosphatase, pNPP = yellow.

i) Reading Results - By using a spectrophotometer, spectrofluorometer, or electrochemical device, the results can be read and recorded. T

Question 18

ELISA has been used for detection of antibodies produced in response to human infection by Borrelia burgdorferi (that causesLyme disease) - If a patient tested antibody-positive for Lyme disease by ELISA – Which of the following test can confirm infection?

Answer 18

Western blot

Question 19

What are the different types of ELISA?

Answer 19

1. Direct ELISA- 1 primary antibody

2.Indirect ELISA- 1 primary and 1 secondary antibody

3.Sandwich ELISA- a capture antibody, 1 primary and 1 secondary antibody

4. Competitive ELISA- 2 primary antibodies with inhibitor antigen