Invitrogen™ Qubit™ Protein and Protein Broad Range (BR) Assay Kits

Catalog No.	Assay	Quantitation Range	Sufficient For
Q33211	Qubit Protein Assay	12.5 μg/mL to 5 mg/mL	100 Reactions
Q33212	Qubit Protein Assay	12.5 μg/mL to 5 mg/mL	500 Reactions
A50668	Qubit Protein Broad Range Assay	0.1 to 20 mg/mL	100 Reactions
A50669	Qubit Protein Broad Range Assay	0.1 to 20 mg/mL	500 Reactions

Catalog No. Q33211 Supplier Invitrogen™ Supplier No. Q33211

Description

Requires

Qubit 1.0 or 2.0 Fluorometer or other fluorometer and 500μL thin-walled PCR tubes

Achieve sensitive detection of protein, and low protein-to-protein variation, with the Qubit Protein and Protein BR Assay Kits for fluorescence-based protein quantitation assays.

Quantify protein quickly and accurately with two unique Qubit protein quantification kits: the Qubit Protein Assay, which quantifies 12.5 μg/mL to 5 mg/mL of protein, and the Qubit Protein BR Assay Kit, which quantifies 0.1 to 20 mg/mL of protein. Both assays are compatible with many common contaminants, reducing reagents, salts, and nucleic acids. The Protein BR Assay Kit is also tolerant of detergents.

Qubit Protein Assay Kit
The Qubit Protein Assay Kit is designed specifically for use with any Qubit fluorometer. While using between 1 and 20 μL of sample, this assay can quantify samples ranging from 12.5 μg/mL to 5 mg/mL with low protein-to-protein variation.

The Qubit Protein Assay Kit is highly selective for proteins and is also highly accurate in the presence of reducing reagents, but not in the presence of large amounts of detergent. Common contaminants, such as reducing reagents (e.g., DTT, β-mercaptoethanol), salts, free nucleotides, amino acids, solvents, and DNA (but not detergents), are well tolerated in the assay. Slight protocol modifications are required for other contaminants.

The kit provides concentrated assay reagent, dilution buffer, and pre-diluted BSA standards. Simply dilute the reagent using the buffer provided, add your sample (any volume between 1 μL and 20 μL is acceptable), and read the concentration.

Which product should you choose for fluorometric protein quantitation?
• 1–20 samples: use this Qubit Protein Assay Kit with the Qubit Fluorometer
• 20–2000 samples: use the Quant-iT Protein Assay Kit with a microplate reader

Qubit Protein BR Assay Kit
The Qubit Protein BR Assay Kit provides a quick, easy, and reliable method for determining the protein concentration of purified proteins, lysates, serum, plasma, or complex protein mixtures using a Qubit 4 Fluorometer. This protein kit can deliver accurate protein quantitation across a broad range (0.1–20 mg/mL), enabling most samples to be used neat (undiluted) and eliminating the guess work and dilution steps that accompany traditional protein quantitation methods. The assay is compatible with many common contaminants, such as detergents, reducing reagents (e.g., DTT, β-mercaptoethanol), salts, and nucleic acids.

Key features of the Qubit Protein BR Assay Kit include:
• Broad dynamic range—0.1 to 20 mg/mL, measure samples without dilution
• Two-point standard curve—eliminate time-consuming standard preparation
• Automatic concentration determination—receive protein concentration instantaneously; no need for offline calculations
• Compatible—accurate in the presence of reducing reagents, detergents, and other common buffer components

The Qubit Protein BR Assay Kit comes with two calibration standards, Protein BR Assay Buffer, and Protein BR Assay Reagent. Simply add 150–160 μL of Protein BR Assay Buffer to 10 or 20 μL of your sample, followed by 30 μL of Protein BR Assay Reagent. Incubate for 10 minutes at room temperature. Then insert the sample into a Qubit 4 instrument and read the concentration.

Notes
• The Qubit Protein Assay Kit can be used with Qubit 1.0, Qubit 2.0, Qubit 3, and Qubit 4 fluorometers
• The Qubit Protein BR Assay Kit is a fluorescence-based assay intended for use only with the Qubit 4 Fluorometer
• 500 μL thin-walled clear PCR tubes (Cat. No. Q32856) are required but not included in the Protein Assay and BR Assay Kits

Order Info

Shipping Condition: Room temperature

Specifications

• Assay: Qubit Protein Assay
• Detection Method: Fluorescence
• Product Line: Qubit
• Product Type: Protein Quantification Assay
• Sufficient For: 100 Reactions
• For Use With (Equipment): Qubit Fluorometer, Microplate Reader
• Quantitation Range: 12.5 μg/mL to 5 mg/mL
• Quantity: 100 Assays
• Shipping Condition: Room Temperature

Frequently Asked Questions (FAQs)

How does the accuracy and sensitivity of the Qubit quantitation assays using the Qubit fluorometer compare to a microplate reader?

The accuracy and sensitivity of the Qubit quantitation assays are the same as that of a microplate reader. This was a requirement during product development. The detection limits for each Qubit kit can be found on the corresponding product manual, which can be found by searching our website by keyword or catalog number.

Can the Qubit kits give an indication of sample quality in nucleic acid samples?

No. The Qubit DNA and RNA kits only quantify the amount of either DNA or RNA in the sample. The Qubit fluorometer cannot take absorbance readings to provide a A260/A280 ratio or detect protein in nucleic acid samples. This can be done with the NanoDrop instrument. If your sample contains protein or other contaminants that can affect the assay, it should be further purified.

If your sample may contain both DNA and RNA, one may use either (or both) the DNA and RNA Qubit kits and compare with samples treated with either RNase or DNase to get an accurate determination of DNA or RNA, respectively.

Can I use the Quant-iT and Qubit Kits with other fluorometers?

All Quant-iT and Qubit kits are compatible with all fluorometers and microplate readers that have the appropriate light sources and filters. You won't have access to the algorithm in the Qubit fluorometer for generating the standard curve provided by the instrument, instead, you must make a few dilutions of the highest standard DNA or RNA (Standard #2) in the Qubit kits to generate a standard curve with multiple data points.

Can I use the original Quant-iT Kits with the Qubit Fluorometer?

No, we do not recommend this. Some of the dyes in the original Quant-iT kits (those NOT listed as “for use with the Qubit fluorometer”) are not compatible with the Qubit Fluorometer. In addition, the new Quant-iT kits (labeled as “for use with the Qubit Fluorometer”) have standards formulated to be compatible with the Qubit fluorometer internal algorithms for the respective assays. The Qubit Fluorometer-compatible kits are also less expensive per assay if you are processing fewer than 20 samples at a time.

Is 1X RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1mM EDTA, pH 7.4) compatible with the Qubit Protein Assay (Cat. No. Q33211/Q33212)?

The concentration of detergents in 1x RIPA buffer is quite high, therefore, we do not recommend that you use this kit with samples in RIPA. The Qubit Protein Assay has a tolerance for detergents present only in very low amounts. Please see the user guide for the tolerance of this assay for detergents here. The Qubit Protein Broad Range (BR) Assay Kit (A50668) has a much higher tolerance for detergents, e.g. RIPA buffer can be used undiluted. Please see this link.

How can I use the Qubit Protein Broad Range Assay (Cat. No. A50668) on the Qubit 4 Fluorometer, with WiFi (Cat. No. Q33238)?

To be able to use the Qubit Protein Broad Range Assay (Cat. No. A50668) on the Qubit 4 Fluorometer (Cat. No Q33238) you would need to install the latest firmware on the fluorometer. Please find the latest Qubit 4 firmware (v2.19) on the following webpage: https://downloads.thermofisher.com/Qubit4/v2.19/Qubit4_update_v2.19.pak Save the firmware update on a USB stick and upload it to the Qubit 4 Fluorometer. Once the firmware update has been installed, you will be able to see the Protein BR assay on the Qubit 4 Fluorometer.

Is the Quant-iT Protein Assay Kit (Cat. No. Q33210) a high-throughput version of Qubit Protein and Protein Broad Range Assay Kits?

Though the Quant-iT Protein Assay Kit is indicated for high-throughput application, it is completely separate from the Qubit Protein and Protein Broad Range Assay Kits, not another version of them.

Are the reagents in the Qubit Protein and Protein Broad Range Assay Kits and those from the Quant-iT Protein Assay Kit (Cat. No. Q33210) the same/interchangeable?

No, the Quant-it and Qubit reagents use different dyes and therefore are not interchangeable.

How do I get "Protein Broad Range" to appear in the Qubit 4 Fluorometer “Protein” menu required to read the standards and samples for the Qubit Protein BR Assay Kit (Cat. Nos. A50668, Q33211, Q33212, A50669)?

To access “Protein Broad Range” in the “Protein” menu of your Qubit 4 instrument, as instructed in the Qubit Protein BR Assay Kit (Cat. Nos. A50668, Q33211, Q33212, A50669) instructions, the instrument will require the latest firmware update (v2.19 or v1.8.0 for the Qubit Flex). You can check which version you currently have on your device by clicking on "Settings" from the Home screen, and then on "About Instrument".

To update the software:

Download the latest firmware update from the Qubit Fluorometers Technical Resources page.
Follow the instructions for updating your Qubit instrument in the firmware Release notes or in the Qubit 4 Fluorometer User Guide (page 53).

After the update has been completed, the option "Protein Broad Range" should become available.

Does the Qubit Protein Assay work well in the presence of detergents?

The Qubit Protein Assay is compatible with very small amounts of detergent. See “Contaminants Tolerated by the Qubit Protein Assay” Table 2 on page 6 of the Qubit Protein Assay Kit product manual (https://tools.thermofisher.com/content/sfs/manuals/Qubit_Protein_Assay_UG.pdf) for specific amounts.

We are using the Qubit Protein Assay Kit (Cat. No. Q33211) with the Qubit 4 Fluorometer. Is this method compatible with the European Pharmacopoeia, chapter 2.5.33?

The Qubit Protein Assay Kit is not described in the European Pharmacopoeia, chapter 2.5.33, i.e., the 7 methods described there are different from the method used for Qubit.

I am getting inaccurate results/poor reproducibility with the Qubit Protein Assay Kit. Can you offer some tips?

Here are possible causes and solutions:

- The kit has expired or has been stored incorrectly: When properly stored, the components of the Qubit Protein Assay Kit should be stable for at least 6 months. Upon receipt, the kit can be stored at 4 degrees C. Components A and B can be stored at ambient temperature and Components C-E can be stored at ≤4 degrees C. Protect Component A from light. High degradation of the BSA standard 2 and 3 will result in a decrease in signal and a “Standards Incorrect” error warning upon calibration. Replace the kit.
- Old calibration data was used: Best practice is to prepare fresh calibration standards at the same time as the samples to take into account any changes in assay conditions.
- Incorrect tubes were used: Use the recommended Qubit Assay Tubes (Cat. No. Q32856). Other 0.5 mL thin-walled PCR tubes may work as well, but performance is not guaranteed. Avoid opaque tubes, as these will block the light path.
- Tubes contain bubbles or particulates that are scattering the light: Pipette gently to avoid the introduction of bubbles. Spin down tubes before measuring to remove bubbles or particulates. Spin down samples to remove particulates before adding an aliquot to the Qubit working solution.
- Inaccurate pipetting: The Qubit assay will accept 1-20 µL of sample, but pipetting very low volumes, especially 1-2 µL is typically very inaccurate, especially with viscous samples. If possible, pipette at least 5 µL for more consistent results.
- The temperature of the assay is changing: Make sure that the Component B buffer is at ambient temperature before use and avoid leaving the samples in the Qubit instrument or near an exhaust fan or other heat source that would warm up the samples.
- Contamination in buffer causing high background: High buffer contamination will show up as an increase in the relative fluorescence (RFU) of the background, measured with the standard tube 1 blank, and eventually will trigger a “Standards Incorrect” error warning on calibration. Replace the kit.
- Sample buffer contains detergents: Qubit protein assay is a detergent-based assay, utilizing an environmentally sensitive dye that fluoresces in the presence of detergents; therefore, only very low concentrations of additional detergents are tolerated in the assay, as listed in Table 2 in the manual (https://tools.thermofisher.com/content/sfs/manuals/Qubit_Protein_Assay_UG.pdf).
- Sample buffer contains other components that are affecting the assay: The Qubit protein assay is generally tolerant of reducing reagents, salts, free nucleotides, amino acids, DNA, and solvents. Table 2 in the manual (https://tools.thermofisher.com/content/sfs/manuals/Qubit_Protein_Assay_UG.pdf) lists acceptable concentrations for many common contaminants. If you have a contaminant you think is affecting the quantitation, then prepare duplicate standard tubes, and spike the contaminant into one set of tubes and run them as samples. If the effect is not too substantial, then spike the buffer into the standards when performing the calibration to account for buffer composition effects.

I would like to use the Quant-iT, Qubit or NanoOrange protein assay kit for quantitation of peptides. Will it work?

The lowest protein size limit for these reagents has not been determined, although proteins as small as 6000 Da have been accurately quantitated. Quantitation accuracy of small peptides would likely be variable and dependent on the composition of the peptide. We would recommend using the CBQCA Protein Quantitation Kit for quantitation of small peptides. For quantitating peptide digest mixtures for mass spectrometry applications, we recommend using the Quantitative Colorimetric Peptide Assay (Cat. No. 23275) or Quantitative Fluorometric Peptide Assay (Cat. No. 23290).

Can the Quant-iT or Qubit protein assays be used in a solution containing protein and liposomes?

We do not recommend that you use the Quant-iT Protein Reagent or Qubit Protein Reagent to quantify proteins in the presence of any detergents, surfactants, lipids or other chemicals that can either displace the dye from a hydrophobic domain, disrupt lipid structure, or add a lipophilic/hydrophobic componentto the solution. The dye is environmentally sensitive; when it binds to hydrophobic pockets/domains, inserts into liposome lipid layers, or is dissolved in organic solvents, its fluorescence output increases relative to its fluorescence in aqueous solutions, potentially providing a higher background. Of course, this assumes that the liposome is not composed of anything that can quench fluorescence.

You may use the dye to quantitate purified protein and possibly a pure liposome sample (assuming that the solution the dye is dispersed in does not disrupt liposome structure), but it would be exceedingly difficult to quantify either as a mixed population.

What are the fluorescent protein assays you offer and how do they differ? Which one should I choose for my samples?

The Quant-iT and Qubit protein assays are easy and accurate assays for the quantitation of protein samples ranging from 12.5 µg⁄mL to 5 mg⁄mL. These assays are highly selective for protein and exhibit very little protein-to-protein variation. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. The Qubit Protein Assay Kit is designed specifically for use with the Qubit Fluorometers, while the Quant-iT Protein Assay is designed to be used both on the Qubit fluorometer or other fluorometers and fluorescence plate readers. 

The NanoOrange Protein Quantitation Kit is a very sensitive and easy assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 10 µg/mL. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. This fluorescent dye is suitable for use with spectrofluorometers and microplate readers. 

The CBQCA Protein Quantitation Kit is a very sensitive assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 150 µg/mL. CBQCA covalently modifies glutamine, asparagine and primary amines and requires cyanide for the reaction. This assay is tolerant of detergents, but amines, ammonium ions and reducing agents should be avoided. CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.

The EZQ Protein Quantitation Kit provides accurate quantitation of protein samples in the range of 20 µg/mL to 5 mg/mL. The assay can be performed in the presence of detergents, urea, reducing agents, salts, solvents, dyes, and most other contaminants and is generally intended for samples prepared for 1D and 2D gel electrophoresis. 1 µL of each sample is spotted onto filter paper placed inside a specially-designed 96-well microplate, contaminants are washed away, and then the remaining bound proteins are stained with our proprietary fluorescent dye. The paper is then analyzed on a microplate reader or a laser scanner.

All the above assay kits come with either concentrated assay reagent and dilution buffer or a pre-diluted quantitation reagent and protein standards. The EZQ Protein Quantitation Kit also comes with a specially-designed 96-well microplate and filter paper that fits inside this microplate.

How can I determine protein concentration in buffers containing imidazole, pH 7.0?

We offer a Quant-iT Protein Assay Kit (Cat. No. Q33210), which is more sensitive than standard absorbance-based assays and can quantitate proteins from 0.25-5 µg. The signal is unaffected by many common contaminants, such as DTT, beta-mercaptoethanol, amino acids, and DNA. Imidazole at a final concentration below 1.25 mM is acceptable. Above that concentration, the imidazole begins to interfere with the assay. Please note, imidazole does absorb at 280 nm, and the absorbance varies with concentration. So to be perfectly accurate, each eluted fraction should be blanked against its elution buffer.