PURPOSE

This procedure describes the determination of a relative total protein concentration using a colorimetric protein assay developed by Bio-Rad Laboratories – “Bio-Rad DC Protein Assay”.

SCOPE

This method applies to samples that require total protein analysis at DSML Enterprises, Inc. with the minimum protein concentration of 25 mg/L (25 mg/mL).

DEFINITIONS

1.     BSA - Bovine Serum Albumin

2.     RTPC - Relative Total Protein Concentration

In this method BSA is used as a relative standard, therefore unless chromophore formation of BSA is similar to the protein being analyzed, only relative protein values maybe obtained.

3.     DC - Detergent Compatible

4.     RO – Reverse Osmosis

5.     SDS - Sodium Dodecyl Sulfate

6.     WR – Working Reagent

7.     PCS – Positive Control Standard.

PCS is a known concentration protein standard that’s analyzed if it were an unknown in order to verify that the procedure is followed correctly and the method works as expected.

 

RESPONSIBILITIES

It is the responsibility of the individual performing the analysis to perform testing of the samples.

SAFETY PRECAUTIONS

Use general safety precautions when working with chemicals. Refer to the MSDS for each product for specific information.

MATERIALS AND EQUIPMENT

Have the following equipment available:

a)             Agilent 8453 UV-Visible Spectrophotometer (model# G1103A) with Multicell Transport (model# G1120A) and Advanced UV-Visible ChemStation Software or equivalent.

b)             Eppendorf Repeater® Plus Pipette with 500 mL, 5 mL and 50 mL Combitips® Plus.

c)             Disposable methacrylate cells, 10 mm (Fisher# 14-385-996 or equivalent). For measurement in the wavelength range 400-1100 nm

d)             Disposable Polyethylene Cuvette Cap Square (Fisher# 14-385-999 or equivalent)

e)             Culture Tubes (12x75 mm) with Cap (Fisher #14-956-3C or equivalent)

f)               Transfer Pipettes, Polypropylene, 5mL (Fisher Scientific or equivalent)

g)             Flat Top Microcentrifuge Tubes, 1.5 mL Polypropylene (Fisher #05-408-10 or equivalent)

h)             Lint Free Wipes (KimwipesŇ or equivalent)

 

Reagents and Standards:

i)               Bio-Rad DC Protein Assay Reagent Package (Bio-Rad #500-0116) contains:

i)               Reagent A (Bio-Rad #500-0113)

ii)              Reagent B (Bio-Rad #500-0114)

iii)             Reagent S (Bio-Rad #500-0115)

j)               Pre- Diluted Protein Assay Standards Set: BSA Fraction V (Pierce #23208 or equivalent)

k)             2 mg/mL Albumin Standard (Pierce #23210 or equivalent)

l)               Reverse Osmosis water, reagent grade, generated at DSML Enterprises, Inc.

m)            Isopropyl Alcohol (IPA), Optima Grade (Fisher Scientific or equivalent)

 

PROCEDURE

NOTE: All standards, reagents, and samples should be warmed up to room temperature before executing the procedure below.

2.              Preparation of 1,000 mg/L BSA PCS

a)             Pipet 3,000 mL of 2 mg/mL Albumin Standard into appropriately labeled culture test tube.

b)             Add 3,000 mL of RO Water to the tube, cap it and vortex well.

c)             Aliquot 200 mL of prepared PCS into 30 appropriately labeled microcentrifuge tubes.

d)             Store at –15oC for up to one year.

e)             Once removed from freezer and opened, pre-aliquot PCS cannot be reused.

3.              Preparation of Working Reagent A´ (necessary only if detergent is present in samples)

a)             Add 20 mL of Reagent S (5% SDS solution) to each milliliter of Reagent A, and mix well in a test tube.

b)             If a precipitate forms, warm the solution and vortex. Do not pipet the undissolved precipitate, as this will likely plug the tip of the pipet, thereby altering the volume of reagent that is added to the standard or sample.

 

4.              Generation of Standard Curve

A standard curve should be prepared each time the assay is performed. The following nine BSA standards are used to generate the standard curve: 0, 25, 125, 250, 500, 750, 1000, 1500, and 2000 mg/L. Only 25 mg/L standard will require preparation, the other ones can be found in the set. The dilution matrix is presented the table below:

UV Cuvette Label

Volume and Source of BSA Standard

Volume of RO Water

Final BSA Concentration

1A

0 mL

50 mL

0 mg/L

1B

0 mL

50 mL

0 mg/L

2A

10 mL of 125 mg/L

40 mL

25 mg/L

2B

10 mL of 125 mg/L

40 mL

25 mg/L

3A

50 mL of 125 mg/L

0 mL

125 mg/L

3B

50 mL of 125 mg/L

0 mL

125 mg/L

4A

50 mL of 250 mg/L

0 mL

250 mg/L

4B

50 mL of 250 mg/L

0 mL

250 mg/L

5A

50 mL of 500 mg/L

0 mL

500 mg/L

5B

50 mL of 500 mg/L

0 mL

500 mg/L

6A

50 mL of 750 mg/L

0 mL

750 mg/L

6B

50 mL of 750 mg/L

0 mL

750 mg/L

7A

50 mL of 1000 mg/L

0 mL

1000 mg/L

7B

50 mL of 1000 mg/L

0 mL

1000 mg/L

8A

50 mL of 1500 mg/L

0 mL

1500 mg/L

8B

50 mL of 1500 mg/L

0 mL

1500 mg/L

9A

50 mL of 2000 mg/L

0 mL

2000 mg/L

9B

50 mL of 2000 mg/L

0 mL

2000 mg/L

 

a)             Execute Assay Protocol as described in PROCEDURE (6) reading the prepared samples on the spectrophotometer as standards.

NOTE: The standards and diluent should be added directly into UV cuvettes.

b)             Calibration Table, Spectra, and Curve should resemble the ones in APPENDIX I.

c)             Correlation Coefficient must be greater or equal to 0.99 (R2 ≥ 0.99); otherwise, the standards should be prepared and assayed again.

5.              Sample Testing

NOTE: Positive Control Standard (PCS)

                                    i)                PCS has to be assayed and tested with every set of samples as if it were an unknown (refer to PROCEDURE (6)).

                                   ii)                The protein concentration should fall within 900 – 1100 mg/L (±10% of target concentration); otherwise, run will be invalidated and samples must be prepared and tested again.

a)             Visually examine the sample.  If there are visible particulates, centrifuge the sample, and assay the supernatant.

b)             If the sample’s protein concentration is expected to be between 50 – 2000 mg/L, no sample preparation is required. Just simply follow Assay Protocol as indicated in PROCEDURE (6) reading samples on the spectrophotometer or microplate reader as unknowns.

c)             If samples’ protein concentration exceeds 2,000 mg/L:

i)               Perform 10 fold serial dilution with RO water so that measured concentration fits within the range of the standard curve

ii)              Test diluted sample according to Assay Protocol as instructed in PROCEDURE (6) reading samples on the spectrophotometer as unknowns.

iii)              

6.              Assay Protocols

Standard (Spectrophotometer)

a)             Setup Spectrophotometer (refer to QC-116)

i)               Turn both lamps on.

ii)              Perform Verification and Diagnostics

iii)             Configure the quantitative method to read absorbance @ 655 nm, and calibrate using linear offset method.

iv)            Zero the spectrophotometer on the cuvette filled only with RO water.

b)             Place UV cuvettes in the rack.

c)             Pipet 50 mL of each standard and unknown sample into UV cuvettes.

d)             Using Repeater® Pipette with 5 mL Combitip® add 250 mL of WR A’ into each cuvette, then gently shake the rack.

e)             Using Repeater® Pipette  - 50 mL Combitip® rapidly add 2.0 mL of Reagent B into each cuvette, cap them, and vortex immediately.

f)               Incubate samples for 15 minutes at ambient temperature.

 

Microplate Reader

NOTE: For operating instructions on Microplate Reader refer to AN-077

a)             Open protocol Bio-Rad DC.ppr. If not present create protocol with the following parameters:

i)               Mode:              Endpoint

ii)              Wavelength:    750 nm

iii)             Automix:          On; 10 seconds before the first read.

iv)            Pathcheck:      Off

v)              

b)             Place UV cuvettes in the rack.

c)             Pipet 10 mL of each standard and unknown into a microtiter plate.

d)             Using Multichannel Pipette add 25 mL of reagent A or A’ into each well.

e)             Using Multichannel Pipette add 200 mL reagent B.

f)               Cover microplate with film then place it onto the Microplate Reader’s tray and shake for 15 second. If bubbles form, pop them with a clean, dry pipet tip.

g)             Incubate samples for 15 minutes at ambient temperature.

h)             Remove the cover, then

 

 

 

 

CALCULATIONS

None (Performed by the software).

RESULTS

1.     Results and calibration data will be printed out by the software.

i)                

TIME REQUIREMENTS

1.     The time required to prepare and run standards for Bio-Rad DC assay (including incubation) is approximately 35 min.

2.     The time required to analyze samples will vary.

 

 

APPENDICES/REFERENCES

1.     APPENDIX I: Examples of Calibration Table, Spectra, and Calibration Curve.

2.     QC-116, “Operation and Verification of the Agilent 8453 Spectrophotometer”.

3.     AN-077, “Operation of the  SPECTRAmax 340PC384 Microplate Reader and SOFTmax Pro software.

 

APPENDIX I

 

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