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:
- UPPA-I solution (75µl) was added and mixed with the samples.
- Mixtures were incubated 2–3 minutes at room temperature.
- UPPA-II solution (75µl) was added and samples were centrifuged for 5 minutes on high speed.
- The tubes were drained and the pellets resuspended in 40µl water + 10µl reagent I (copper solution).
- 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.
- 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:
- 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.
- A 245µl aliquot of the reagent A+B was then added to microcentrifuge tubes followed by 5µl of each BSA dilution.
- 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.
- Fluorescence was measured using a Cytofluor® II fluorescent plate reader (excitation @ 485nm, emission @ 580nm, gain setting @ 40).
Previous results (Figure 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 2) demonstrate that diluting the Steady-Glo® and Bright-Glo™ Reagents to 0.1X resulted in acceptable linearity between protein concentration and absorbance.
Figure 2. BSA standard curve generated using 0.1X Steady-Glo® Reagent or 0.1X Bright-Glo™ Reagent (or water control) with the NI™ Protein Assay.
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 3) 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.
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.