RC DC Protein Assay
Instruction Manual
Catalog # 500-0119 500-0120 500-0121 500-0122
For Technical Service
Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Section 1 |
Introduction |
The RC DC Protein Assay is a colorimetric assay for protein quantitation with all the functionality of the original DC Protein Assay. This assay is based on the Lowry1 assay but has been modified to be reducing agent compatible (RC) as well as detergent compatible (DC).
Section 2 Product Description
RC Reagents Package, includes
•RC Reagent I (250 ml)
•RC Reagent II (250 ml)
(Sufficient for 500 standard assays or 2,000 microfuge tube assays)
RC Reagent I contains UPPA-I
RC Reagent II contains UPPA-II
UPPA is a trademark of Geno Technology, Inc.
Section 3 Reagent Compatibility
The listed reagents were tested and found to be compatible with the RC DC Protein Assay. The presence of one or more of these substances may change the response of the protein to the assay reagents. Thus the protein standard should always be prepared in the same buffer as the protein sample.
Reagents |
One Wash |
Two Washes (Optional) |
Dithiothreitol (DTT) |
100 mM |
350 mM |
Tributylphosphine (TBP) |
2 mM |
- |
β-mercaptoethanol |
5% |
10% |
Sequential Extraction Buffer 2♦ |
Not Compatible |
Full Strength |
Sequential Extraction Buffer 3♦♦ |
Not Compatible |
Full Strength |
Laemmli Buffer |
|
|
(with 5% β-mercaptoethanol) |
Full Strength |
- |
CHAPS |
2% |
- |
Tween 20* |
2% |
- |
Triton X-100** |
2% |
- |
EDTA |
100 mM |
- |
Imidazole |
500 mM |
- |
Tris, pH 8.4 |
500 mM |
- |
NaOH |
2.5 M |
- |
*Tween is a registered trademark of ICI Americas, Inc. **Triton is a registered trademark of Rohm and Haas.
♦40 mM Tris, 8 M urea, 4% (w/v) CHAPS, 0.2% (w/v) Bio-Lyte 3/10 ampholyte, 2 mM TBP (Catalog #163-2103)
♦♦40 mM Tris, 5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (w/v) SB 3-10, 0.2% (w/v) Bio-Lyte 3/10 ampholyte, 2 mM TBP (Catalog #163-2104)
1
Section 4 Assay Instructions
Standard Assay Protocol (5 ml)
1Add 20 µl of DC Reagent S to each 1 ml of DC Reagent A that will be needed for the run. This solution is referred to as Reagent A´. Each standard or sample assayed will require 510 µl of Reagent A´.
(Reagent A´ is stable for one week even though precipitate will form after one day. If precipitate forms, warm the solution and vortex. Do not pipet the undissolved precipitate as this will likely plug the tip of the pipet and alter the volume of Reagent A´ added to the sample.)
2Prepare 3-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml protein. A standard curve should be prepared each time the assay is performed.
(For best results, the standards should always be prepared in the same buffer as the sample.)
3Pipet 100 µl of standards and samples into clean, dry test tubes.
4Add 500 µl RC Reagent I into each tube, vortex. Incubate the tubes for 1 minute at room temperature.
5Add 500 µl RC Reagent II into each tube, vortex. Centrifuge the tubes at 15,000xg for 3-5 minutes.
6Discard the supernatant by inverting the tubes on clean, absorbent tissue paper. Allow the liquid to drain completely from the tubes.
7Add 510 µl Reagent A´ to each tube, vortex. Incubate tubes at room temperature for 5 minutes, or until precipitate is completely dissolved. Vortex before proceeding to the next step.
8Add 4 ml of DC Reagent B to each tube and vortex immediately. Incubate at room temperature for 15 minutes.
9After the 15 minutes incubation, absorbances can be read at 750 nm. The absorbances will be stable for at least 1 hour.
Microfuge Tube Assay Protocol (1.5 ml)
1Add 5 µl of DC Reagent S to each 250 µl of DC Reagent A that will be needed for the run. This solution is referred to as Reagent A´. Each standard or sample assayed will require 127 µl of Reagent A´.
(Reagent A´ is stable for one week even though precipitate will form after one day. If precipitate forms, warm the solution and vortex. Do not pipet the undissolved precipitate as this will likely plug the tip of the pipet and alter the volume of Reagent A´ added to the sample.)
2Prepare 3-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml protein. A standard curve should be prepared each time the assay is preformed.
(For best results, the standards should always be prepared in the same buffer as the sample.)
2
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