Fluorescence-based protein quantification detection methods provide superior sensitivity which means you use less of your protein samples for quantitation and have more samples available for your experiment. These fluorescence assays are also useful when colorimetric assays cannot be used due to interfering color in the samples. For these assays, few steps are required, and timing is not critical so the assays can be adapted for automated handling in high-throughput applications. The fluorescence signal can be detected using a fluorometer or microplate reader.
Choose the right fluorescent assay for your sample
| Qubit Protein BR Assay | Qubit Protein Assay | NanoOrange Protein Quantification Assay | CBQCA Plus Protein Quantification Assay | EZQ Protein Quantification Assay | |
|---|---|---|---|---|---|
| Best for | Rapid quantitation of small number of samples | Quick quantitation of small number of samples | Dilute samples or samples with limited volume | Dilute samples or samples with limited volume containing detergents or lipids | High-throughput analysis through solid-phase assay format |
| Assay range (sample volume) | 100 µg/ml to 20 mg/ml (10–20 µL) | 12.5 µg/ml to 5 mg/ml (1–20 µL) | 10 ng/mL to 10 µg/mL (up to 10 µL) | 50 ng/mL to 150 µg/mL | 20 µg/mL to 5 mg/mL (1 µL) |
| Assay incubation time and temperature | 10 min at RT | 15 min at RT | 10 min at 90–95°C | 1 hr at RT | 1 hr at RT |
| Detection wavelength (nm) | 470/570 | 470/570 | 470/570 | 465/550 | 280 and 450/618 |
| Required equipment | Qubit 4 Fluorometer | Qubit Fluorometer | Fluorometer, or fluorescence microplate reader | Fluorometer, or fluorescence microplate reader | Fluorescence microplate reader, or laser-based scanning systems, or CCD imaging systems |
| Compatible reagents | Detergents, reducing agents, salts | Reducing agents, salts | Reducing agents, urea, salts | Detergents, reducing agents (below 100 µM) | Reducing agents, detergents, urea |
| Incompatible reagents | Glycine | Detergents | Detergents | Ammonium salts, amines such as Tris or glycine | |
| Standard curve | 2-point | 3-point | 5-point | 5- to 7-point | 5-point |
| Signal duration | 1 hr | 3 hr | 6 hr | 5 hr | --- |
| Mechanism of action | Reacts with primary amines found in proteins (N-terminus and epsilon amines found in lysines) | Binds to detergent coating on proteins and hydrophobic regions of proteins; the unbound dye is nonfluorescent | Binds to detergent coating on proteins and hydrophobic regions of proteins; the unbound dye is nonfluorescent | Reacts with primary amine groups on proteins in the presence of cyanide or thiols; the unreacted dye is nonfluorescent | Binds electrostatically to basic amino acids, supplemented by additional hydrophobic interactions |
| Cat. No. | A50668 (100 assays) A50669 (50 assays) | Q33211 (100 assays) Q33212 (500 assays) | N6666 | A66522 | R33200 |
| Qubit Protein BR Assay | Qubit Protein Assay | NanoOrange Protein Quantification Assay | CBQCA Plus Protein Quantification Assay | EZQ Protein Quantification Assay | |
|---|---|---|---|---|---|
| Best for | Rapid quantitation of small number of samples | Quick quantitation of small number of samples | Dilute samples or samples with limited volume | Dilute samples or samples with limited volume containing detergents or lipids | High-throughput analysis through solid-phase assay format |
| Assay range (sample volume) | 100 µg/ml to 20 mg/ml (10–20 µL) | 12.5 µg/ml to 5 mg/ml (1–20 µL) | 10 ng/mL to 10 µg/mL (up to 10 µL) | 50 ng/mL to 150 µg/mL | 20 µg/mL to 5 mg/mL (1 µL) |
| Assay incubation time and temperature | 10 min at RT | 15 min at RT | 10 min at 90–95°C | 1 hr at RT | 1 hr at RT |
| Detection wavelength (nm) | 470/570 | 470/570 | 470/570 | 465/550 | 280 and 450/618 |
| Required equipment | Qubit 4 Fluorometer | Qubit Fluorometer | Fluorometer, or fluorescence microplate reader | Fluorometer, or fluorescence microplate reader | Fluorescence microplate reader, or laser-based scanning systems, or CCD imaging systems |
| Compatible reagents | Detergents, reducing agents, salts | Reducing agents, salts | Reducing agents, urea, salts | Detergents, reducing agents (below 100 µM) | Reducing agents, detergents, urea |
| Incompatible reagents | Glycine | Detergents | Detergents | Ammonium salts, amines such as Tris or glycine | |
| Standard curve | 2-point | 3-point | 5-point | 5- to 7-point | 5-point |
| Signal duration | 1 hr | 3 hr | 6 hr | 5 hr | --- |
| Mechanism of action | Reacts with primary amines found in proteins (N-terminus and epsilon amines found in lysines) | Binds to detergent coating on proteins and hydrophobic regions of proteins; the unbound dye is nonfluorescent | Binds to detergent coating on proteins and hydrophobic regions of proteins; the unbound dye is nonfluorescent | Reacts with primary amine groups on proteins in the presence of cyanide or thiols; the unreacted dye is nonfluorescent | Binds electrostatically to basic amino acids, supplemented by additional hydrophobic interactions |
| Cat. No. | A50668 (100 assays) A50669 (50 assays) | Q33211 (100 assays) Q33212 (500 assays) | N6666 | A66522 | R33200 |
Learn more about Microplate Assays for Protein Quantitation
Resources
Pierce Protein methods – an encyclopedia of articles about protein analysis
Protein assay technical guide – tools and reagents for improved quantitation of total or specific proteins.
Protein assay compatibility table – summarizes compatible substances for several popular protein assays.
Protein Analysis Learning Center – resources for different protein analysis techniques
Protein Biology Application Notes – journal-style articles based on research applications and proof-of-concept experiments
Qubit Fluorometers – quantitate the amount of DNA, RNA, or protein with a simple interface and built-in calculator
Microplate readers – measure fluorescence, absorbance, luminescence, or time-resolved fluorescence
For Research Use Only. Not for use in diagnostic procedures.