Automating the Bradford Method with a Pipetting Robot

The Bradford method is a colorimetric assay, which requires the creation of a standard curve, in order to quantify the concentration of protein of unknown samples. Conducted by Reform Biologics, a company based in Cambridge, Massachusetts, this study explores the automation of a Bradford assay (which is microplate based) for reproducibility.

The study assesses the ability of a pipetting robot to consistently achieve a high coefficient of determination (R2). The Andrew Alliance pipetting robot generates three independent standard curves, and attains an R2 value greater than or equal to 0.998. This value demonstrates the robot’s capability to automate microplate assays with high precision.

The Bradford assay is designed for total protein quantitation, and is a colorimetric method. Protein is bound in acidic medium by Coomassie dye, which causes an absorption shift from 465 nm to 595 nm. The concentration of protein is measured by comparing an unknown sample’s absorbance at 595 nm, with the absorbance values of a calibration curve prepared using known protein concentrations. The Bradford method was chosen as a representative colorimetric assay for automation with the Andrew Alliance pipetting robot.

Materials

  • Andrew 1000G liquid handling robot: Andrew Alliance (Geneva, Switzerland)
  • Bovine gamma globulin standard, 2.0 mg/mL: Thermo Fisher Scientific (Rockford, IL)
  • Pierce Coomassie Plus (Bradford) assay reagent: Thermo Fisher Scientific (Rockford, IL)
  • 0.01 M phosphate buffered saline (NaCl 0.138 M); pH 7.4: Sigma-Aldrich (St. Louis, MO)
  • Nunc MicroWell 96-well microplate: Thermo Fisher Scientific (Rockford, IL)
  • Pipetman Classic pipettes: Gilson Inc (Middleton, WI)
  • Synergy HT plate reader: BioTek (Winooski, VT)

Methods

Coomassie Plus was the assay reagent in the performance of the Bradford method, according to the manufacturer’s directions. Standard solutions of different BGG concentrations were generated by diluting bovine gamma globulin (BGG) standard with phosphate buffered saline (PBS). These standard solutions, with a PBS blank, were then pipetted into a 96-well microplate in triplicate, followed by the Coomassie Plus reagent.

The next steps were transferring the loaded microplate to a plate reader, where it was shaken for 30 seconds, then incubated at ambient temperature for 10 minutes, and the absorbance measured at 595 nm. A standard curve was generated by plotting the average blank-corrected 595 nm absorbance measurements for each BGG standard, against the known concentration in µg/mL units. The data was fitted with a quadratic curve (y= ax2 + bx + c) for the concentration range of 125 µg/mL to 2000 µg/ mL, and the R2 value subsequently determined using Microsoft Excel software.

The Andrew 1000G executed a single protocol in order to prepare standard BGG solutions in 2 mL conical tubes. The solutions were pipetted onto a 96-well microplate and then Coomassie Plus reagent added to each well. The slow pipetting speed remained consistent in all steps involving a BGG solution. The feature of the “air top cushion” was used to subsequently transfer to the microplate 10 µL of each BGG solution. The precise and repetitive pipetting mode was used to rapidly add 300 µL of Coomassie Plus reagent to each of the microplate’s wells. To generate three independent Bradford assay standard curves, this protocol was run three times.

Figure 1. Andrew robot loading microplate while performing Bradford assay

Andrew Lab protocol statistics

Figure 2. Andrew Lab protocol statistics

Standard curves from three independent Bradford assays performed by the Andrew Robot. Error bars represent standard deviation of solution run in triplicate. Solid line is quadratic fit to data for each run.

Figure 3. Standard curves from three independent Bradford assays performed by the Andrew Robot. Error bars represent standard deviation of solution run in triplicate. Solid line is quadratic fit to data for each run.

Table 1. Mean absorbance measurements of standard solutions for three pipetting runs with standard deviation (SD) and coefficient of variation (CV).

BGG (µg/mL) Run 1 Run 2 Run 3 Manual Run
Average SD CV (%) Average SD CV (%) Average SD CV (%) Average SD CV (%)
2000 0.843 0.015 1.73 0.880 0.015 1.71 0.857 0.024 2.77 0.844 0.068 8.00
1500 0.752 0.032 4.30 0.766 0.021 2.73 0.750 0.018 2.36 0.738 0.017 2.31
1000 0.582 0.030 5.21 0.562 0.013 2.30 0.579 0.018 3.11 0.512 0.029 5.75
750 0.432 0.014 3.24 0.461 0.026 5.56 0.469 0.025 5.40 0.434 0.012 2.75
500 0.305 0.005 1.65 0.298 0.012 4.10 0.287 0.013 4.41 0.241 0.007 2.92
250 0.136 0.016 11.70 0.139 0.014 9.82 0.146 0.009 6.14 0.090 0.008 9.32
125 0.064 0.007 11.03 0.061 0.005 8.13 0.063 0.004 5.75 0.030 0.011 36.72

 

Table 2. Summary of three pipetting runs.

  Quadratic fit parameters R2
a b c
Run 1 -0.1497 0.7344 -0.0207 0.9986
Run 2 -0.1347 0.7209 -0.0191 0.9991
Run 3 -0.1475 0.7329 -0.0162 0.9980
Manual Run -0.0494 0.6940 -0.1210 0.9950

 

Conclusions

  • To perform microplate based assays, such as the Bradford assay in this procedure, the Andrew robot can be used, with extremely high repeatability.
  • By completing repetitive pipetting steps, the Andrew robot frees up scientist time, by increasing both laboratory workflow and efficiency.
  • The Andrew robot is able to offer consistent assay run times, which generates vastly more accurate project planning.

About Andrew Alliance S.A.

Andrew Alliance is an independent, privately financed company, based in Geneva, Boston and Paris. The company was created in March 2011.

Andrew Alliance is dedicated to advance science by working with scientists to create a new class of easy-to-use robots and connected devices that take repeatability, performance, and efficiency of laboratory experiments to the level required by 21st-century biology.

Start with meeting customer needs, end with customer feedback.

Andrew Alliance delivers solutions that are focused on customer needs, both today and in the future. Our products are manufactured to the highest standards, using a range of carefully selected, proven, and sustainable technologies, that ensure both high performance and reliability. We actively seek continuous customer feedback, in order to guarantee the best possible design outcomes.


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Last updated: Jul 22, 2019 at 8:01 AM

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