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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. |
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:
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.
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.
