Bio-Rad RC DC Protein Assay User Manual

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
Lowry1assay 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
Sequential Extraction Buffer 3
Laemmli Buffer

(with 5% β-mercaptoethanol) Full Strength -

CHAPS 2% -

*

Tween 20
Triton X-100
EDTA 100 mM ­Imidazole 500 mM -
Tris, pH 8.4 500 mM ­NaOH 2.5 M -

**

♦

Not Compatible Full Strength

♦♦

Not Compatible Full Strength

2% -
2% -

*

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)

1

Add 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.)

2

Prepare 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.)

3 Pipet 100 µl of standards and samples into clean, dry test tubes.
4 Add 500 µl RC Reagent I into each tube, vortex. Incubate the tubes for 1

minute at room temperature.

5 Add 500 µl RC Reagent II into each tube, vortex. Centrifuge the tubes at

15,000xg for 3-5 minutes.

6 Discard the supernatant by inverting the tubes on clean, absorbent tissue

paper. Allow the liquid to drain completely from the tubes.

7 Add 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.

8 Add 4 ml of DC Reagent B to each tube and vortex immediately. Incubate at

room temperature for 15 minutes.

9 After 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)

1 Add 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.)

2 Prepare 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