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.
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 before use by inversion.
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 in column 1, …) to the optical plate.
Cover plate with aluminum foil and mix on a plate shaker at medium speed for 1 minute.
Incubate plate at 25C for 5 minutes. Samples should turn green.
Using a plate reader, measure the absorbance of the samples at 660 nm.
Analyze results.
Subtract the absorbance of blank samples (e.g., BSA standard = 0 mg/uL) from all other samples (”background subtracted absorbance”).
Prepare the standard curve by plotting the background subtracted absorbance vs. concentration for each BSA sample.
Fit the standard curve by linear regression.
For each dilution series of each sample, choose a dilution in the 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