Assignment Detail:- 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
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