Our team aimed to develop a library of oral drugs consisting of inhibitory peptides effective against Malaria caused by Plasmodium falciparum. These drugs consist of short peptides that have been designed to inhibit specific interactions between Plasmodium falciparum and human erythrocyte proteins that are engineered into circular, plant-based proteins called cyclotides.
In order to measure the efficacy of our drug, we need to experimentally verify that the extent of binding between the host and parasite protein is reduced in the presence of our drug. For this purpose, we have designed an assay called the Ni-NTA protein binding assay, which can be found on our Experiments page. In this assay, protein concentrations are measured to show that the amount of host protein bound to the parasite protein decreases in the presence of our drug. The Bicinchoninic Acid Assay is a quantitative assay, designed by Paul K. Smith[1] at the Pierce Chemical Company that measures the amount of protein in a given sample. The absorbance value at 562 nm is a measure of the amount of protein in the sample. This absorbance is given in Absorbance units. BSA is a protein that is used to calibrate the absorbance values of the spectrophotometer before taking readings during the experiment. In order to get comparable readings across spectrophotometers, we have developed a standardisation protocol for the same.
While developing drugs against Malaria, the IC50 assay serves as an important assay. This assay can be found on our Experiments page. It measures the concentration of the proposed drug that is required to inhibit a particular process or molecule by 50 percent. It uses a fluorescent DNA binding dye - SYBR Green1 to measure the DNA content of the parasite present in the sample. However, the fluorescence readings that we obtain are in arbitrary units. The arbitrary values obtained are non reproducible and vary greatly between different instruments, which is not suitable for an engineering field like synthetic biology. Hence, to be able to get comparable data we have written a standardisation protocol which uses known concentrations of DNA to calculate the fluorescence values for a given amount of SYBR Green1 dye.
IC50 Standardization Protocol
Abstract
In order to get comparable results across instruments while performing the assay, you may follow this standardisation protocol. By the end of this protocol, a graph of fluorescence readings vs DNA concentration is obtained. This graph can be used to compare the order of fluorometric readings that are obtained from different instruments.
Protocol
Take 100 µL of RPMI in the wells of 96 well-plate.
Prepare a stock solution of 200 ng of DNA concentration in TAE (Tris-acetate-EDTA) solution (volume = 100 µL).
Perform serial dilution (2-fold)
Take 100 µL of the stock solution and transfer it into a well containing 100 µL TAE (the DNA concentration of the solution is now reduced to 100 ng).
Take 100 µL of solution from well 1 and transfer it into well 2 containing 100 µL TAE (the DNA concentration of the solution is now reduced to 50 ng).
Take 100 µL of solution from well 2 and transfer it into well 3 containing 100 µL TAE (the DNA concentration of the solution is now reduced to 25 ng).
Take 100 µL of solution from well 3 and transfer it into well 4 containing 100 µL TAE (the DNA concentration of the solution is now reduced to 12.5 ng).
Take 100 µL of solution from well 4 and transfer it into well 5 containing 100 µL TAE (the DNA concentration of the solution is now reduced to 6.25 ng).
Take 100 µL of solution from well 5 and transfer it into well 6 containing 100 µL TAE (the DNA concentration of the solution is now reduced to 3.125 ng).
Take 100 µL of solution from well 6 and transfer it into well 7 containing 100 µL TAE (the DNA concentration of the solution is now reduced to 1.5625 ng).
Take 100 µL of solution from well 7 and transfer it into well 8 containing 100 µL TAE (the DNA concentration of the solution is now reduced to 0.78125 ng).
Blank (background signal control)
Take 100 µL of TAE in well 9 (no DNA).
Prepare a 10X stock of SYBR Green 1 + Lysis Buffer (volume = 2500 µL).
Add 11.25 µL of 10X SYBR Green 1 + Lysis buffer from the stock to each well.
Mix and incubate for 45 minutes at room temperature in dark.
Take fluorometer readings in Thermo Fisher VarioskanFlash
Select the lane
Select fluorometry based assessment
Set up mode to:
Excitation
495 nm
Emission
520 nm
Select type of plate i.e. Flat Bottom/Conical/Round Bottom
In order to compare the absorbance outputs reported by different teams, it is necessary for each team to create a standard fluorescence curve. For this purpose, the following standardisation protocol may be followed to get comparable data. By the end of this protocol, a graph of absorbance readings vs protein concentration is obtained. This graph can be used to compare the order of absorbance readings that are obtained from different instruments.
Protocol
A. Preparation of bovine serum albumin (BSA) standards
Decide on a working range of protein concentrations. This protocol is for a standard range of 10 to 100 µg/mL.
Prepare dilutions of BSA using the same diluent/buffer as the samples.
From the BSA stock solution (1000 µg/mL) take 5 µL and transfer it to a vial. Add 45 µL of the diluent to this.
From the BSA stock solution take 4.5 µL and transfer it to a vial. Add 45.5 µL of the diluent to this.
From the BSA stock solution take 4 µL and transfer it to a vial. Add 46 µL of the diluent to this.
From the BSA stock solution take 3.5 µL and transfer it to a vail. Add 46.5 µL of the diluent to this.
From the BSA stock solution take 3 µL and transfer it to a vial. Add 47 µL of the diluent to this.
From the BSA stock solution take 2.5 µL and transfer it to a vial. Add 47.5 µL of the diluent to this.
From the BSA stock solution take 2 µL and transfer it to a vial. Add 48 µL of the diluent to this.
From the BSA stock solution take 1.5 µL and transfer it to a vial. Add 48.5 µL of the diluent to this.
From the BSA stock solution take 1 µL and transfer it to a vial. Add 49 µL of the diluent to this.
From the BSA stock solution take 0.5 µL and transfer it to a vial. Add 49.5 µL of the diluent to this.
In one vial add 50 µL of the diluent. This will be our blank.
Calculate the total volume of WR required by using the given formula:
(# protein solutions) × (# replicates) × (volume of WR per sample) = total volume WR required
For our standardization:
11 × 3 × 100 µL = 3300 µL or 3.3 mL WR required
Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B)
For our standardization:
5 mL of Reagent A + 0.1 mL of Reagent B would be be combined
C. Absorbance Measurement
Pipette 10 µL of each protein solution replicates into a microplate well.
Add 100 µL of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds.
Cover plate and incubate at 37°C for 25 minutes.
Cool plate to room temperature.
Measure the absorbance at or near 562 nm on a plate reader.
1: Smith, P. E., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M., ... & Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical biochemistry, 150(1), 76-85.
