Protein Determination Warburg-Christian Method

Document History

Replaces SS-PWC01. Document number changed to conform to current Operating Procedure numbering system.

1. OBJECTIVE
To standardize a procedure for the determination of protein content by Warburg-Christian at Sigma-Aldrich, St. Louis, MO.

2. SCOPE
This procedure applies to all products that require the determination of protein by Warburg-Christian method.

3. DEFINITIONS
Purified water – water from a deionizing system, resistivity > or = 18 MΩ•cm @ 25°C

4. DISCUSSION
N/A

5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.

6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7. PROCEDURE

7.1 CONDITIONS:
T = 25°C, A260nm and A280nm, Light Path = 1 cm

7.2 METHOD:
Spectrophotometric

7.3 REAGENTS:
Protein Sample Solution (Sample)

Prepare a solution containing approximately 0.3 mg protein/ml of sample in suitable buffer or purified water.

7.4 TEST METHOD
Read the A260nm and the A280nm of sample in a suitable cuvette versus a suitable blank. The A280nm must be greater than the A260nm in order to obtain valid results. The presence of NAD or NADP will affect the A260nm and A280nm of proteins and thus, when necessary, must be corrected for in the calculations.

8. CALCULATIONS

mg protein/ml = [(1.31 x A280) - (0.57 x A260)] x dilution factor

% protein = (1.31 x A280) - (0.57 x A260) x 100
mg solid/ml


*mg protein/ml = [(5 x A280) - A260] x dilution factor
4.635


*correction for NAD or NADP

9. REFERENCES & ATTACHMENTS
Warburg, O. and Christian W. (1941) Biochem. Z. 310, 384-421

10. APPROVAL

  Print Name Sign Name Title Date
Prepared by: Nathan Vogel Nathan Vogel Originator 10/19/04
Approved by: Rodney Burbach Rodney Burbach Supervisor, Analytical Services 10/20/04
Approved by: Gene McNaughton Gene McNaughton Quality Assurance 10/21/04

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