Qubit™ Protein Assay Kit, 100 Assays - FAQs

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.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.

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.

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.

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.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

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

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Quantitation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.