Qubit Protein Assays

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	Qubit Protein BR Assay	Qubit Protein Assay
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)	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.	A50668 (100 assays) A50669 (500 assays)	Q33211 (100 assays) Q33212 (500 assays)

*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.

Qubit Protein BR Assay workflow

Qubit Protein Assay workflow

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%.

Accurate determination of protein load from complex protein mixtures

The Qubit Protein BR Assay and a standard Bradford assay were used to determine the protein concentration of lysates from several mammalian cell types: 293T, A549, HepG2, HeLa, and iPSCs. Lysates were separated on an Invitrogen NuPAGE 4–12% Bis-Tris Mini Protein Gel and labeled with No-Stain Protein Labeling Reagent. (A) Gel image was acquired on the iBright FL1500 Imaging System and (B) normalization factors were determined using Invitrogen iBright Analysis Software. The CV of the Qubit Protein BR Assay was appreciably lower than that of the Bradford assay.

Buffer compatibility table

Qubit protein assay compatibility with common buffer components

Contaminant	Qubit Protein BR Assay	Qubit Protein Assay
Contaminant	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