Focus: Protein determination in Promega buffers
Quantitating Protein in Promega Lysis Buffers and Steady-Glo® and
Bright-Glo™ Luciferase Reagents
The
ability to perform protein quantitation assays in six Promega luciferase buffers was
determined using two commercially available assays. We recommend diluting Steady-Glo® (Cat.# E2520) and
Bright-Glo™ (Cat.# E2620) Luciferase
Assay Reagents to 0.1X. For cell lysis buffers, we recommend a buffer concentration of
0.1X or 1X, depending upon the specific assay used. |
By Natalie Betz, Ph.D.
Promega Corporation
Introduction
Knowing the protein concentration of cell extracts to be used in reporter assays is
important, as it allows for normalization of reporter signal to total protein amount to be
determined.
The purpose of these experiments was to identify whether two different protein assays
could be used to determine protein concentration with Promega's cell lysis buffers as well
as reconstituted Steady-Glo® (Cat.# E2520)
and Bright-Glo™ (Cat.# E2620) Luciferase
Assay Reagents(*). These luciferase reagents
include components for cell lysis and provide for a homogeneous luciferase reporter assay.
Homogeneous reporter assays are assays in which quantitation directly from cells grown in
multiwell plates is performed. Preparation of lysates is not required.
Four Promega lysis buffers were tested: Reporter Lysis
Buffer (RLB; Cat.# E3971), Cell Culture Lysis Reagent (CCLR; Cat.# E1531), Passive Lysis Buffer (PLB; Cat.# E1941)
and Glo Lysis Buffer (GLB; Cat.# E2661),
each at 1X and 0.1X concentration. Two commercially available protein quantitation assays
were tested: NI™ (Non-Interfering) protein assay by GenoTechnology, Inc. (Catalog
#786-005) and NanoOrange® protein quantitation kit by Molecular Probes
(Catalog #N-6666).
Promega's RLB, CCLR, PLB and GLB buffers contain detergents and reducing and chelating
agents. The NI™ protein assay works in the presence of reducing agents, chelating
agents, detergents and amines, as well as many other compounds and buffers. This product
includes "universal protein precipitating agents" (UPPA™) that remove the
proteins from the interfering agents present in solution. The assay is based on the
specific binding of copper ions to the peptide backbone of proteins. The color density is
inversely related to the amount of protein present. The manufacturer states that the assay
is linear between 0.5-50µg for the standard and microassay protocols.
The NanoOrange® kit contains a primary reagent that is virtually
nonfluorescent in aqueous solution but undergoes a dramatic enhancement in fluorescence
upon interaction with proteins. When bound to proteins in the provided diluent, the
reagent exhibits a broad excitation peak at 470-490nm and a broad emission peak at
570-590nm. The assay can be used with standard fluorometers, minifluorometers and
fluorescent microplate readers. The manufacturer states the sensitivity of the assay to be
between 10ng/ml-10µg/ml; the assay is supposed to be insensitive to reducing agents and
nucleic acids.
Methods and Results
RLB, CCLR, PLB and GLB were diluted to either 1X or 0.1X using Nuclease-Free Water. The
Steady-Glo® and Bright-Glo™ substrates were resuspended in the buffers
provided with each reagent. Serial dilutions of BSA (Factor V from Sigma) were prepared in
each of the lysis buffers, luciferase assay reagents or in water, from a 100mg/ml
stock.
NI™ Protein Assay: BSA dilutions were prepared
at 0, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0 and 20.0µg per 5µl. An aliquot (5µl) of each was
then processed to determine protein concentration according to the manufacturer's
protocol:
1. UPPA-I solution (75µl) was added and mixed with the samples.
2. Mixtures were incubated 2-3 minutes at room temperature.
3. UPPA-II solution (75µl) was added and samples were centrifuged for 5 minutes on
high speed.
4. The tubes were drained and the pellets resuspended in 40µl water + 10µl reagent I
(copper solution).
5. Following a 3-minute incubation at room temperature, 100µl of Reagent II (100 parts
A + 1 part B) were added to each sample, mixtures transferred to a flat-bottom, 96 well
plate and incubated at room temperature for 20 minutes.
6. Absorbance at 480nm was recorded using a SpectraMax® 250 microplate
reader.
NanoOrange® Protein Quantitation Assay: Dilutions of BSA
were made as above and processed according to the manufacturer's
protocol:
1. Reagent A+B was prepared by diluting Reagent B 1:10 with water and then diluting
Reagent A 1:500 using the Reagent B dilution.
2. A 245µl aliquot of the reagent A+B was then added to microcentrifuge tubes followed
by 5µl of each BSA dilution.
3. Samples were incubated for 10 minutes at 90°C, allowed to cool to room temperature
for 20 minutes, then transferred to a white Dynex flat-bottom, 96 well plate.
4. Fluorescence was measured using a Cytofluor® II fluorescent plate reader
(excitation @ 485nm, emission @ 580nm, gain setting @ 40).
Previous results (1) demonstrated the NI™ protein
assay to be compatible with 1X RLB, CCLR, PLB, and GLB lysis buffers. This protein assay
was tested further with 1X Steady-Glo® and Bright-Glo™ Luciferase
Reagents. The results of this experiment (data not shown) demonstrated that the Steady-Glo®
and Bright-Glo™ reagents gave less than optimal results at 1X concentrations, with
high background readings and no linearity with protein concentration. Perhaps something in
the Steady-Glo® and Bright-Glo™ Reagents interferes with either the
protein precipitation step or the color development step. [Note: absorbance decreases with
increasing protein concentration with this assay].
To determine whether diluting the Steady-Glo® and Bright-Glo™ Reagents
improved the compatibility with the NI™ protein assay, these reagents were diluted
1:10 and a BSA standard curve was generated. The results (Figure 1) demonstrate that diluting
the Steady-Glo® and Bright-Glo™ Reagents to 0.1X resulted in acceptable
linearity between protein concentration and absorbance.
We next investigated whether the various lysis buffers and luciferase reagents were
compatible with the NanoOrange® protein quantitation assay. This commercial
assay kit is easy to use, especially in high-throughput applications as compared to the
NI™ protein assay. BSA standard curves (Figure 2) were generated in 1X or 0.1X
RLB, CCLR, PLB, GLB, Steady-Glo® Reagent and Bright-Glo™ Reagent. For
this assay system, fluorescence increases with increasing protein concentration, as seen
in Figure 2 for the water control. At 1X concentrations, all lysis buffers and luciferase
reagents did not appear to be compatible with this protein kit. The background readings
were high and relative fluorescence was not linear with respect to protein concentration
(data not shown).
However, at 0.1X each of the lysis buffers and luciferase reagents exhibited reduced
background levels and increased linearity (Figure 2). The background levels for
GLB and Steady-Glo® and Bright-Glo™ Reagents were still relatively high,
but a linear relationship between fluorescence and protein concentration was observable.
Table 1. Recommended Buffer Concentration
for Performing Protein Quantitation. Recommendations are for Promega lysis
buffers and luciferase reagents as indicated. |
Buffer |
NI™ Protein Assay |
NanoOrange®
Protein Quantitation Kit |
RLB |
1X |
0.1X |
CCLR |
1X |
0.1X |
PLB |
1X |
0.1X |
GLB |
1X |
0.1X |
Steady-Glo® Reagent |
0.1X |
0.1X |
Bright-Glo™ Reagent |
0.1X |
0.1X |
Notes:
RLB = Reporter Lysis Buffer (Cat.# E3971)
CCLR = Cell Culture Lysis Reagent (Cat.# E1531)
PLB = Passive Lysis Buffer (Cat.# E1941)
GLB = Glo Lysis Buffer (Cat.# E2661)
Conclusions
To determine protein concentration using the NanoOrange® fluorescent
protein assay (Molecular Probes) in any of the four lysis buffers or luciferase reagents,
samples must first be diluted 1:10 with water prior to analysis. For the NI™ protein
assay (GenoTechnology) in either the Steady-Glo® or Bright-Glo™ Reagent,
samples also must be diluted 1:10 with water; this assay can be used with any of the four
lysis buffers undiluted. For accurate protein quantitation using these or any other assay
kits, the standard curve must be generated in the same dilution of lysis buffer or
luciferase reagent.
When performing protein assays in experiments that include the Steady-Glo®
or Bright-Glo™ Reagents, cells must first be washed once with 1X PBS. Then
luciferase reagent is added to an equal volume of 1X PBS, which is added in turn to the
cells. Do not add the reagent to standard culture media as sera will interfere with the
protein assay. Alternatively, cells can be lysed using a lysis buffer, and an aliquot of
the lysate is then used for protein determination as described above.
Reference
- Betz, N. (2000) Protein determination in Promega Cell Lysis Buffers using a
noninterfering protein assay, Promega eNotes (www.promega.com/enotes/applications/
ap0021_tabs.htm).
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
Steady-Glo is a trademark of Promega Corporation and is registered with the U.S.
Patent and Trademark Office. Bright-Glo is a trademark of Promega Corporation.
Cytofluor is a registered trademark of Millipore Corporation. NanoOrange is a
registered trademark of Molecular Probes. NI is a trademark of GenoTechnology. SpectraMAX
is a registered trademark of Molecular Devices Corporation.
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