Getting Started
Pierce660 is a quick (5 min) colorimetric method for total protein quantitation. Compared to other quantitation assays, we find Pierce660 to be simple and reproducible, with a wide dynamic range (50-2000 ug/mL), and robust to buffer composition, including detergents and reducing agents.
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Storage Conditions | Link |
Reagents | ||||||
Pierce660 Reagent | Pierce™ 660nm Protein Assay Reagent | Thermo Scientific | 22660 | $176.65 | 4C to 30C | [link] |
BSA Protein Standard | Pierce™ Bovine Serum Albumin Standard Ampules, 2 mg/mL | Thermo Scientific | 23209 | $81.65 | 4C to 30C | [link] |
Consumables | ||||||
96-well optical plate | Microplate, 96 well, PS, U-bottom, clear | Greiner | 650101 | $156.14 | 4C to 30C | [link] |
Equipment | ||||||
Plate Reader | BioTek Cytation 5 Cell Imaging Multimode Reader | Agilent | CYT5MFAWSN | Must request quote | 4C to 30C | [link] |
Protocol
Prepare a standard curve within the assay’s working range (125 ug / mL to 2000 ug / mL). Remember to dilute the BSA stock in the same buffer used for your sample. The standards can be stored at -20C for future assays.
Concentration | Volume of BSA Stock (2 mg/mL) | Volume of Buffer |
2 mg/mL | 200 uL | 0 uL |
1.5 mg/mL | 150 uL | 50 uL |
1 mg/mL | 100 uL | 100 uL |
0.75 mg/mL | 75 uL | 125 uL |
0.50 mg/mL | 50 uL | 150 uL |
0.25 mg/mL | 25 uL | 175 uL |
0.125 mg/mL | 12.5 uL | 187.5 uL |
0 mg/mL | 0 | 200 uL |
Prepare a dilution series of your samples in the same buffer.
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Mix Pierce660 Reagent well by inverting the bottle before use.
Array 150 uL of Pierce660 Reagent on a 96-well optical plate.
Add 10 uL of each sample (BSA standard series and sample concentration series) column-wise (e.g., BSA standard in Column 12, Sample 1 series in column 1, …) to the optical plate.
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Cover your plate with aluminum foil and mix on a plate shaker at medium speed for 1 minute.
Incubate your plate at 25C / 5 min. Samples should turn from brown to green.
Using a plate reader, measure the absorbance of the samples at 660 nm.
Analyze results.
Subtract the absorbance of blank samples (i.e., BSA standard = 0 mg / uL) from all other samples (”background subtracted absorbance”).
Plot the standard curve by plotting the background subtracted absorbance vs. concentration for each BSA standard. Fit a line to your standard curve.
For each sample dilution series, choose a well with a background subtracted absorbance in the linear range of the standard curve.
Using the linear fit of your standard curve, calculate the concentration of the sample.
Resources and References
Pierce660 Manual.pdf375.4KB