Oct 18, 2019
Bradford Assay with RotiQuant (ROTH)
- iGEM Dusseldorf1
- 1Heinrich-Heine Universität Düsseldorf
Protocol Citation: iGEM Dusseldorf 2019. Bradford Assay with RotiQuant (ROTH). protocols.io https://dx.doi.org/10.17504/protocols.io.8gghttw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 18, 2019
Last Modified: October 18, 2019
Protocol Integer ID: 28904
Keywords: quantification of protein, protein, assay, comparing measured absorbance, measured absorbance, rotiquant
Abstract
Assay for quantification of protein by comparing measured Absorbance at 595 nm to bovine serum albumine standard
Materials
Bovine serum albumin (concentrated >200 μg/ml)
Roti-Quant 5x (ROTH)
96 Well Plate
Plate Reader
Troubleshooting
Standard measurements
Dilute your BSA stock solution to 200µg/ml in the same buffer used for your solution of interest.
Dilute the Roti-Quant 5x reagent at a rate of 2:7,5. For 15 ml solution, this means 4 ml Roti-Quant added to 11 ml of Water.
Create 200 µl each of standard concentrations according to the following table:
| Concentration [µg/ml] | Volume of stock needed | Volume of solution buffer needed | |
| 0 | 0 | 200 | |
| 20 | 20 | 180 | |
| 40 | 40 | 160 | |
| 60 | 60 | 140 | |
| 80 | 80 | 120 | |
| 100 | 100 | 100 |
Work in Triplicates. Pipette 50µl of the standards into the 96-Well plate
Add 200 µl of the diluted Bradford reagent to each well.
Incubate for 5 minutes at room temperature
Measure the OD595 for each well. Create a regression line from the averages of each concentrations' measurements
Protein concentration measurements
Dilute your solution of interest 1:20 or 1:40, depending on expected protein levels.
Work in triplicates and use blanks. Pipette 50 µl of each dilution into a well on the 96-Well plate.
When working with solutions containing pigments, prepare additional wells to which NO Bradford reagent will be added
Add 200 µl of the diluted Bradford reagent to each well. For the pigment controls, add 200 µl of solution buffer instead. Optimally, use a multi-pipette to reduce the time needed for adding the reagent.
Incubate for 5 minutes at room temperature
Measure the OD595 for each well.
When processing the data, correct the measurements for the blanks. When pigments are present in the solution, correct the OD595 by subtracting the OD595 of the pigment controls from the appropriate measurements.
Determine the amount of protein by putting the achieved OD595 of your solution of interest into the regression formula and multiplying by the dilution factor.