Reference no: EM133106187

BC3102 Molecular Basis of Disease - James Cook University

Quantitation of Specific Proteins Using an ELISA

Learning objective 1: Demonstrate accurate and precise pipetting skills;

Learning objective 2: Demonstrate an understanding of quantitative assays based on antibody specificity;

Learning objective 3: Demonstrate an understanding of the use of standards and titrations in quantitation; Identify the concentration of a protein in an unknown sample.

Objective: To quantify the amount of tropomyosin in two unknown samples.

Reagents and Equipment
• 1 ml standard protein A (recombinant tropomyosin)
• 2 unknown samples that may contain tropomyosin
• Coating buffer (0.016M Na2CO3, 0.034M NaHCO3, pH 9.6)
• 96-well flat bottom High Binding ELISA plate
• Washing solution 1 x PBS/Tween (1x PBS with 0.05% Tween 20), 2L
• Distilled water
• Blocking solution (5% skim milk in PBS with 0.05% Tween 20)
• Rat monoclonal anti-tropomyosin antibody (1:1000) (Primary Antibody)
• Rabbit anti-mouse IgG - HRP (Horse Radish Peroxidase conjugate (1:10,000)
• (Secondary Antibody)
• TMB (tetra methyl benzidene)-substrate
• Stopping solution (1N Hydrochloric acid)
• ELISA plate reader

Procedure

1. Prepare a set of standard dilutions (STD) from the standard antigen stock (rTM) by performing double dilutions in 1.5mL tubes using the carbonate buffer.

2. Prepare a set of 8 standard dilutions (STD1-8). Label them tubes 1-8.

3. Prepare two dilution series of each of the unknown samples (SAM 1 & SAM 2) using carbonate buffer.

4. Once all the dilutions have been prepared, add 100 μL of each of the standards, unknowns, controls and blanks in triplicates or duplicates as indicated in the ELISA map. For the negative controls (NEG CTRL) do not add antigen. Seal the plate with cling wrap and incubate at 28°C for 30 minutes.

5. Discard the antigen solution and block the wells using 250μL of blocking buffer and incubate at room temperature for 20 minutes.

6. Discard the blocking solution and wash the wells with PBS-T (Phosphate buffered saline with 0.05% Tween 20) three times for 2 minutes each.

7. Add 100 μL/well of the primary (anti-tropomyosin ) antibody. For the primary antibody control (P.Ab CTRL), do not add the primary (anti-tropomyosin ) antibody. Reseal the plate and incubate for 1 hour at 28°C on gentle shaking.

8. Discard the primary antibody and wash the plate as described above.

9. Add 100 μL of the secondary antibody to the wells and incubate for 30 minutes at room temperature.

10. Discard the secondary antibody and wash the plate as described but four times.

11. Add 100 μL of the TMB substrate and cover the plate with aluminium foil and develop for 10 minutes or till a blue colouration is formed.
(Note: You MUST wear nitrile gloves during step 11, as TMB is an irritant)

12. Stop the reaction by adding 50 μL of 1N (1M) Hydrochloric acid to all the wells (This step must be carried out in the fumehood)

13. Measure the absorbance at 450 nm on the ELISA reader.

Write up and report

Prepare a standard curve (OD450 vs concentration) for the ELISA readouts and determine the concentrations of tropomyosin in the unknown samples

Part B Questions

1. What is the significance of diluting the unknown samples?

2. Why do we need to maintain a primary antibody control?

3. Why is there no positive control?

4. Why did you use the dilution strategy chosen (serial or individual dilutions)

Attachment:- Molecular Basis of Disease.rar