Protein Determination Modified Lowry Method

1. OBJECTIVE

To standardize a procedure for the determination of protein by modified Lowry.

2. SCOPE

This procedure applies to all products that have a specification for Lowry protein determination without Trichloroacetic Acid (TCA) precipitation.

3. DEFINITIONS

Purified water - from a deionizing system, resistivity > or = 18 MΩ•cm @ 25 °C

4. DISCUSSION

Copper + Protein	Alkaline pH >	Copper-protein complex
	Phenol reagent

5. RESPONSIBILITIES

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

6. SAFETY

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. PROCEDURE

7.1 CONDITIONS:

T = 25 °C, A_750nm, Light path = 1 cm

7.2 METHOD:

Colorimetric

7.3 REAGENTS:

7.3.1 0.85% Sodium Chloride Solution (NaCl) Use Sodium Chloride Solution, 0.85%, Product Number S0817, or prepare 100 mL in purified water using Sodium Chloride, Product Number S9888.

7.3.2 0.01% Lowry Protein Standard (WPS) Prepare 10 mL in Reagent 7.3.1 using Product Number P0914 or a working protein standard prepared per our “Preparation of Working Protein Standard for Use in Protein Determination” (Operating Procedure 10-30-6802).

7.3.3 Modified Lowry Reagent (LOW) Prepare by reconstitution in purified water, per label instructions, a vial of Lowry Reagent, Modified, Product Number L1013 or Product Number L3540 . This solution can be stored for approximately one month or until the solution becomes cloudy.

7.3.4 Protein Sample Solution (Sample) Prepare a solution containing 10-100 mg protein/mL of sample in reagent 7.3.1.

7.3.5 0.33 N Folin & Ciocalteu's Phenol Reagent (F&C) Prepare a solution by performing a 6-fold dilution using 2 N Folin & Ciocalteu's Phenol Reagent, such as Product Number F9252, in purified water. Mix thoroughly.

7.4 TEST METHOD

Inhibition Reaction

Pipette (in milliliters) the following reagents into a suitable containers:

	Test	Std 1	Std 2	Std 3	Std 4	Std 5	Blank
Reagent 7.3.1 (NaCl)	-----	0.90	0.70	0.50	0.30	-----	1.00
Reagent 7.3.2 (WPS)	-----	0.10	0.30	0.50	0.70	1.00	-----
Reagent 7.3.3 (LOW)	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Reagent 7.3.4 (Sample)	1.00	-----	-----	-----	-----	-----	-----

Mix thoroughly by vortexing and incubate for 10 minutes at 25 °C. Incubation time must be at least 10 minutes to allow for the copper solution to react with peptide bonds. This time can be as long as 24 hours if needed.
Then add:

	Test	Std 1	Std 2	Std 3	Std 4	Std 5	Blank
Reagent 7.3.5 (F&C)	0.50	0.50	0.50	0.50	0.50	0.50	0.50

Mix thoroughly by vortexing and incubate for 30 minutes at 25 °C. Incubation time must be at least 30 minutes but no longer than 60 minutes, because color development will continue after 30 minutes. All standards, samples, and blank absorbances must be read on spectrophotometer within a short time of each other.

Transfer the solutions to suitable cuvettes and record the A_750nm for Test, Standards, and Blank using a suitable spectrophotometer.

7.5 CALCULATIONS

ΔA_750nm Standard = A_750nm Standard – A_750nm Blank
Prepare a standard curve by plotting the ΔA_750nm of the Standards vs. mg of protein.
Sample Determination:
ΔA_750nm Test = A_750nm Test - A_750nm Blank
Determine the mg of protein from the Standard Curve.
mg Protein = (mg of protein from the Standard curve) * (df)
df = Dilution factor

% Protein =	(mg Protein) * (100)
	(mg solid/mL)

100 = Conversion to percentage

mg Protein/mL	(mg Protein)
	(mL sample/mL)

100 = Conversion to percentage

8. REFERENCES & ATTACHMENTS

1. Lowry OH, Rosebrough NJ, Farr AL, Randall R. 1951. J. Biol. Chem.(193):265-275.

2. Peterson G. 1979. Analytical Biochemistry.(100):201-220.

9. APPROVAL

	Print Name	Sign Name	Title	Date
Prepared by:	Holly Chapman	Holly Chapman	Originator	11/9/04
Approved by:	Rodney Burbach	Rodney Burbach	Supervisor, Analytical Services	11/10/04
Approved by:	Gene McNaughton	Gene McNaughton	Quality Assurance	11/10/04