BC3102 Molecular Basis of Disease Assignment Help and

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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