Pierce660 Assay

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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.

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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]

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[ ] 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.
- Notes: we do 2x serial dilutions
[ ] 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.
- Notes: adjust your sample volume as needed
[ ] 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.

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Pierce660 Manual.pdf