Rapid, accurate protein quantification
The Qubit Protein Assay Kits are designed to make protein quantification easy while providing accurate quantification. Qubit protein assays require only 10–15 minutes of room temperature incubation, eliminating long incubation periods or exposure to elevated temperatures.
Addressing overlapping concentration ranges, the two kits together are accurate for initial sample concentrations from 12.5 μg/mL to 20 mg/mL and exhibit low protein-to-protein variation. Common contaminants, such as reducing reagents (DTT, β-mercaptoethanol), salts, free nucleotides, amino acids, solvents, DNA, and detergents (in the Qubit Protein BR assay) are well tolerated in the assays. Other contaminants may require slight modifications to the procedure.
Note: While the Qubit Protein Assay runs on both the Qubit 4 and Flex Fluorometers, the Qubit Protein BR Assay runs only on the Qubit 4 model.
Protein quantification assays at a glance
Feature |
||
Appropriate for |
A broad range of protein concentrations and types |
Testing dilute samples, <100 µg/mL |
Compatible platform |
Qubit 4 Fluorometer |
Qubit Flex, Qubit 4 Fluorometer* |
Features |
Simple, rapid assay with 2-point standard curve |
Simple, rapid assay with 3-point standard curve |
Sample volume required |
10 or 20 µL |
1–20 μL |
Compatibility |
Detergents |
Reducing agents (eg. TCEP, DTT) |
Incompabilities |
Glycine |
Detergents (eg, SDS, Tween 20, Triton X-100) |
Initial sample range |
100 µg/mL to 20 mg/mL |
12.5 µg/mL to 5 mg/mL |
Quantitation range |
1–400 µg |
0.25–5 µg |
Cat. Nos. |
*The Qubit Protein Assay is also supported on older Qubit models, Qubit 2 and Qubit 3.
Wide dynamic range
The wide dynamic range of the Qubit Protein assays, in comparison to standard assays such as Bradford assays, enables most samples to be used neat (undiluted). This eliminates the guesswork and dilution steps that accompany traditional protein quantitation methods.
Qubit Protein assays provide a much wider dynamic range than standard protein quantitation assays like Bradford assays. The Qubit Protein assay can accurately quantify proteins down to 12.5 µg/mL, while the Qubit Protein BR assay (compatible with the Qubit 4 Fluorometer) has a quantitation range up to 20,000 µg/mL.
Simple, rapid quantification
Qubit protein assays are easy to perform and set up with just two or three standards to prepare, unlike traditional assays which typically require a 5– to 7–point standard curve for protein quantitation.
Accurate protein quantification
Proteins are diverse in their composition and structure. Differences in amino acid sequence, isoelectric point (pI), secondary structure, and side chains or prosthetic groups can result in variation in the quantitated amount. The Qubit Protein BR Assay provides accurate protein quantitation with low protein-to-protein variability compared to traditional assays such as the Bradford assay.
When loading an electrophoresis gel for western blot analysis to study protein expression, accurate protein loading is essential. In this experiment, the Qubit Protein BR Assay and a traditional Bradford Protein Assay were used to determine a 10-µg protein load for five different cell lysates. No-Stain Protein Label Reagent was used to estimate the total protein lane loads on the iBright FL 1500 Imaging System. The Qubit Protein BR Assay accurately determined the protein concentration of the lysates with a coefficient of variation (CV) of 11% between loads, whereas the Bradford assay exhibited higher variability with a CV of 28%.
Buffer compatibility table
Qubit protein assay compatibility with common buffer components
Contaminant |
Qubit Protein BR Assay |
Qubit Protein Assay |
Compatible concentration in sample |
||
β-Mercaptoethanol |
1 mM |
10 mM |
Acetonitrile |
20% |
|
Ammonium sulfate |
200 mM |
50 mM* |
Bicine |
100 mM |
|
Borate (50 mM pH 8.5) |
Neat |
|
B-PER reagent |
Neat |
|
CHAPS |
5% |
|
Carbonate-bicarbonate |
Neat |
|
DTT |
5 mM |
10 mM* |
DMSO |
10% |
|
EDTA |
50 mM |
10 mM |
Glucose |
1 M |
|
Glycerol |
10% |
10%* |
Guanidine-HCl |
4 M |
|
Imidazole |
200 mM |
12.5 mM |
I-PER |
Neat |
|
Mem-PER |
Neat |
|
MES |
125 mM |
|
MOPS |
100 mM |
|
M-PER |
Neat |
|
Sodium acetate |
100 mM |
|
Sodium azide |
|
10 mM |
Sodium chloride |
5 M |
200 mM* |
NE-PER (CER) |
Neat |
|
NE-PER (NER) |
Neat |
|
NP-40 |
5% |
Ø |
Phosphate-buffer saline (PBS), pH 7.4 |
Neat |
10 mM KPO4, 150 mM NaCl* |
Potassium phosphate, pH 7.4 |
|
5 mM |
PMSF |
1 mM |
|
RIPA |
Neat |
|
SDS |
5% |
0.1%* |
Sucrose |
20% |
500 mM |
T-PER |
Neat |
|
Tricine |
50 mM |
|
Tris-buffer saline (TBS) |
Neat |
|
Tris-glycine, pH 8.0 |
Ø |
|
Tris-glycine SDS, pH 8.3 |
Ø |
|
Tris-HCl |
500 mM |
|
Tris-HEPES SDS, pH 8.0 |
Neat |
|
Triton X-100 |
5% |
Ø |
Tween 20 |
3% |
Ø |
Urea |
3 M |
|
Y-Per |
Ø |
|
GPCR Extraction & Stabilization Reagent |
1:2 |
|
Cell Surface Protein Isolation Kit |
Neat |
|
*An acceptable result, but with some distortion of the standard curve. For best results, add the same amount of contaminant to the standard samples.
Method. The Qubit Protein BR Assay and Qubit Protein Assay were performed according to instructions with samples of 1,000 µg/mL of BSA containing commonly used buffers and contaminants. Assays were performed in triplicate, and RFU values were compared to that of BSA in 0.9% NaCl, 0.05% NaN3. The assay was considered compatible with the tested substance at the indicated concentration if the error in protein concentration estimation caused by the presence of the substance was less than 10%. Concentrations listed refer to the actual concentration in the protein sample. Ø denotes compounds that were not compatible at the lowest concentration tested. Blank fields denote that the compounds were not tested with that assay.
Buffer formulations used in compatibility testing
Buffer |
Formulation |
Sodium carbonate-bicarbonate, pH 9.4 |
0.2 M sodium carbonate-bicarbonate, pH 9.4 |
Phosphate-buffered saline (PBS) |
100 mM sodium phosphate, 150 mM NaCl, pH 7.2 |
RIPA buffer |
25 mM Tris, 150 mM NaCl, 1% DOC, 1% NP-40, 0.1% SDS, pH 7.6 |
Tris-buffered saline (TBS) |
25 mM Tris, 150 mM NaCl, pH 7.4 |
Tris-glycine, pH 8.0 |
25 mM Tris, 192 mM glycine, pH 8.0 |
Tris-glycine-SDS, pH 8.3 |
25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 |
Tris-HEPES-SDS |
100 mM Tris, 100 mM HEPES, 3 mM SDS |
For Research Use Only. Not for use in diagnostic procedures.