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Focus: Protein Determination
Protein Determination in Promega Cell Lysis Buffers Using a Noninterfering Protein
Assay
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| This experiment investigated whether a new protein
determination assay (NI Protein Assay™) was compatible with the various Promega cell lysis buffers. We found this
assay to be compatible with all of the Promega cell lysis buffers. Show Me the Data! |
By Natalie Betz, Ph.D.
Promega Corporation
Introduction
The Non-Interfering Protein Assay™ (Geno
Technology, Inc., Cat. #786-005; 500 standard assays) was developed to be resistant to
interference by reducing agents, chelating agents, detergents, amines, denaturing agents,
sugars, heavy metals and other reagents as well. Basically, the proteins are precipitated
out of solution using proprietary universal protein precipitating agents (UPPA). The
precipitated protein is mixed in an alkaline solution containing copper salt, and the
copper ions bind with the peptide backbone of the protein. The unbound copper ions are
measured by a colorimetric assay, and thus the color density is inversely related to the
amount of protein present. The assay is not affected by protein side chain groups, which
overcomes protein-to-protein variations that exist in other types of assays.
Reducing agents, detergents and chelating agents are found in many of the Promega lysis
buffers, which can complicate routine protein determination. Standard curves using BSA
(bovine serum albumin) were generated using the non-interfering assay with the BSA sample
in water, Reporter Lysis Buffer (RLB, Cat.# E3971), Luciferase Cell Culture Lysis Reagent, 1X* (CCLR; Cat.# E1531), Passive Lysis Buffer (PLB, Cat.# E1941), or Glo Lysis Buffer (GLB, Cat.# E2661; buffer for
use with the Bright-Glo™ (Cat.# E2610)
and Steady-Glo™ (Cat.# E2510) Luciferase Assay Systems*).
Methods and Results
BSA (Sigma Factor V) from a 100mg/ml stock was diluted in water such that 0, 1, 5, 10,
20, 50 or 100µg BSA was present in a 5µl volume. The BSA was then added to 45µl of
water or one of the above lysis buffers already made 1X if not provided as such. Thus the
final sample volume was 50µl, which is the maximal amount for the standard protein assay
using this kit. The protein assay was performed exactly as outlined in the manual provided
with the kit. The 2ml-assay protocol was followed. (There is also a 96 well microplate
format in which the volumes of the reagents are 10% that of the standard assay.) The
following protocol was followed.
Add BSA and water/lysis buffer to 1.5ml microcentrifuge tubes for a final volume of
50µl (5µl BSA + 45µl water/1X lysis buffer). Each reaction was performed in duplicate
(UPPA stands for universal protein precipitating agents). To each tube:
- Add 0.5ml UPPA-I solution, vortex and incubate at room temperature for 3 minutes.
- Add 0.5ml UPPA-II solution, mix by inverting tube and place in microcentrifuge
(maximum speed) for 5 minutes.
- Discard supernatant and let tubes drain for 5-10 minutes.
- Resuspend protein pellets in 0.4ml water + 0.1ml Reagent I copper solution.
- Add 1ml Reagent II (100 parts Color Agent A + 1 part Color Agent B).
- Incubate at room temperature for 15-20 minutes.
- Transfer 200µl of each sample to a Costar® flat-well 96 well plate.
- Read absorbance (480nm) in a SpectraMAX® 250 plate reader.
Averaged results of the duplicate tubes were plotted as protein amount (0-100µg)
against absorbance (480nm). The results can be seen in Figure 1;
the plot of 0-20µg is expanded and shown in the lower panel.
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Figure 1. BSA standard curve. Upper Panel: 0-100µg
protein. Lower Panel:
0-20µg protein. |
In general, the BSA standard curves are quite similar, regardless of whether the assay
was performed in water or one of the four lysis buffers (RLB, CCLR, PLB or GLB). At lower
amounts of protein (i.e., 0-20µg), the differences between the various buffers is more
apparent, but the curves are still linear and could be used to determine the protein
concentration of an unknown sample.
Conclusion
The Geno Technology, Inc., Non-Interfering Protein Assay™ is compatible with all
of the Promega cell lysis buffers tested. When using this assay, the minimal amount of
protein that can be detected with confidence is 5µg in a 50µl volume (i.e., protein
solution of 0.1mg/ml). When generating a BSA standard curve for use in calculating the
protein concentration of an unknown sample, the standard curve should always contain the
same amount (and concentration) of lysis buffer as the sample being measured. Note that
when using this specific protein assay, the maximal amount of protein that can be
confidently measured is approximately 100-200µg. Thus cell lysates may need to be diluted
to obtain accurate readings if they are greater than 20mg/ml.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
Bright-Glo is a trademark of Promega Corporation.
Costar is a registered trademark of Corning, Inc. Non-Interfering Protein Assay is a
trademark of Geno Technology, Inc. SpectraMAX is a registered trademark of Molecular
Devices Corporation.
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