Thermo Fisher Qubit Protein BR Assay User Manual

TECHNICAL NOTE Qubit Protein BR Assay

Qubit Protein BR Assay—fast,
accurate protein quantitation

Protein quantitation is an integral part of many protein
biology workflows and a necessary step before commonly
used techniques such as protein electrophoresis, western
blotting, mass spectrometry, and immunoassays. The
Invitrogen™ Qubit™ Protein BR Assay is a fluorometric
assay that combines accuracy, compatibility, and ease of
use, making protein concentration determination easier
and fas te r.

The Qubit Protein BR Assay is optimized to work with a
wide range of sample concentrations and components.

Add 150 or 160 µL

Add 10 or 20 µL

Std 2Std 1 Sample

Blank

Protein BR
Assay Buer

Add 30 µL

Protein
BR
Reagent

The assay is easy to perform and only requires a 10 minute
incubation at room temperature (RT), eliminating the need
to wait for long incubation periods or expose samples to
elevated temperatures. The assay protocol, seen in Figure
1, is easy to set up with just two standards to prepare,
unlike traditional assays that typically require a 7-point
standard curve for quantitation.

Here we demonstrate the utility of the Qubit Protein BR
Assay and compare it with many well-known assays used
for protein quantitation.

Mix and vortex for 5 sec

Final volume is 200 µL

Step 1: Prepare
assay tubes

Figure 1. Qubit Protein BR Assay protocol.

Step 2: Add Protein
BR Assay Buer

Step 3: Add Protein
BR Reagent

Step 4: Incubate for
10 min at RT

Step 5: Read samples

Key features

0

1,000

2,00 0

3,000

4,000

5,000

6,000

7,000

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 5,000 10,000 15,000 20,000 25,000

RFU

Absorbance

Sample concentration (µg/mL)

Qubit Protein BR Assay

Bradford

BCA

HepG2

HepG2

• Rapid assay with only 2 standards to prepare and
10 min incubation

• Compatible with detergents and reducing agents

• Broad dynamic range, 100–20,000 µg/mL

Broad dynamic range

One of the major advantages of using the Qubit Protein
BR Assay is its broad dynamic range in comparison to
standard colorimetric protein assays. The broad linear
response allows accurate determination of unknown protein
concentrations and provides a higher dynamic range than
other standard protein assays (Figure 2). The Qubit Protein
BR Assay can be used to detect protein concentrations
from 100 to 20,000 µg/mL, allowing most samples to
be used neat (undiluted), eliminating the guesswork and
dilution steps that accompany standard protein quantitation
methods.

Accurate protein determination

The Qubit Protein BR Assay provides accurate protein
quantitation with low protein-to-protein variability as
compared to traditional assays such as the Bradford assay.
Proteins are diverse in their composition and structure, and
dierences in amino acid sequence, isoelectric point (pI),
secondary structure, and side chains or prosthetic groups
can result in variation in the quantitated concentration.

To demonstrate the accuracy and low protein-to-protein
variability of the Qubit Protein BR Assay, several dierent
cell lysates were generated, and total protein concentration
was determined with the Qubit Protein BR Assay and
a Bradford protein assay. Based on the calculated
concentrations, the amount of each lysate containing 10
µg of protein was loaded onto a protein gel. The accuracy
of the total protein loads was evaluated using Invitrogen™
No-Stain™ Protein Labeling Reagent in combination with
lane normalization analysis on an Invitrogen™ iBright™
FL1500 Imaging System. The load variation produced
by the Qubit Protein BR Assay was relatively low, with
a coecient of variation (CV) of 11%, whereas the load
variation produced by the Bradford assay was 2.5 times
higher, with a CV of 28% (Figure 3).

A

Protein BR Assay

Lysate

293T

10 µg protein loads

A549

HepG2

HeLa

iPSC

Bradford assay

293T

A549

HepG2

HeLa

iPSC

a Working range

Qubit Protein BR Assay 100–20,000 µg/mL

BCA Assay 20–2,000 µg/mL

Bradford Assay 125–1,500 µg/mL

Figure 2. Standard curves for protein quantitation assays. Purified
bovine serum albumin (BSA) in 0.9% saline (0–20 mg/mL) was used to
generate standard curves for the Qubit Protein BR Assay (red), Thermo
Scientific
Assays were conducted following the manufacturers’ protocols. The BCA
and Bradford assays were performed in microplate format.

™

Pierce™ BCA Protein Assay (blue), and the Bradford assay (gray).

B

1.4 0

1.2 0

1.0 0

0.80

0.60

0.40

Normalization factor

0.20

0.00

293T

Figure 3. 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
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 the Invitrogen

Analysis Software.

Total lane normalization

CV: 11%

iPSC

A549

HeLa

™

NuPAGE™ 4–12% B i s-Tris

293T

CV: 28%

A549

HeLa

iPSC

™

iBright™