Introduction

The Bradford assay is a colorimetric method for total protein quantitation. Coomassie dye binds protein in acidic medium causing a shift in absorption from 465 nm to 595 nm. Protein concentration is measured by comparing the absorbance at 595 nm of an unknown sample with absorbance values of a calibration curve prepared using known protein concentrations. Here the Bradford method was selected 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

The Bradford method was performed using Coomassie Plus Bradford assay reagent according to the directions provided by the manufacturer. A bovine gamma globulin (BGG) standard was diluted with phosphate buffered saline (PBS) to generate standard solutions of different BGG concentrations. The standard solutions and a PBS blank were then pipetted into a 96-well microplate in triplicate followed by the Coomassie Plus reagent. The loaded microplate was then transferred to a plate reader, shaken for 30 seconds, incubated at ambient temperature for 10 minutes, and absorbance was measured at 595 nm. A standard curve was prepared by plotting the average blank-corrected 595 nm absorbance measurements for each BGG standard against the known concentration in μg/mL units. The data were fit with a quadratic curve (y𝑦= ax2 + bx + c ) for the concentration range of 125 μg/mL to 2000 μg/ mL, and R2 value was determined with Microsoft Excel software.

A single protocol was executed by the Andrew 1000G to prepare standard BGG solutions in 2 mL conical tubes, pipette the solutions onto a 96-well microplate and then add Coomassie Plus reagent to each well. The slow pipetting speed was used in all steps involving a BGG solution. The “air top cushion” feature was used to transfer 10 μL of each BGG solution to the microplate. The repetitive precise pipetting mode was used to quickly add 300 μL of Coomassie Plus reagent to each well of the microplate. The protocol was run three times to generate three independent Bradford assay standard curves.

AN_Automated_Colorimetric

An Automated Colorimetric

Conclusions

• The Andrew robot can be used to perform microplate based assays, such as the Bradford assay shown here, with high repeatability.

• The Andrew robot frees up scientist time by completing repetitive pipetting steps, improving workflow and efficiency in the lab.

• The Andrew robot provides consistent assay run times, facilitating more accurate project planning.

 

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