Standard Operating Procedure
Title: Bradford Assay to measure the concentration of protein in leaf extract
solution
Department: Agronomy
Created by: Whitney Bouma
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Allen Knapp
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview:
This procedure outlines the Bradford method which is used for quantifying the amount of
protein. It is a colorimetric assay that will use a spectrophotometer to construct a standard
curve which will be used to estimate the amount of protein in your sample.
Equipment and reagents necessary:
Equipment:
Spectrophotometer
Glass test tubes
Beaker
Weight boats
Spatula
Standard Disposable Polystyrene Cuvettes
Pipettes
Stir bar
Stir plates
Reagents:
Coomassie Blue G250
Ethanol 95%
Phosphoric acid 85%
Deionized water
Bovine Serum Albumin
Leaf extract samples
Procedure:
Reagent Preparation:
Bradford Reagent:
1. Measure out 100mg of Coomassie Blue G250.
2. Make 500 ml 95% stock solution by measuring 450 mL ethanol and making up
to a volume of 500 ml with DI H2O.
3. Dissolve the Coomassie blue in 50 mL of 95% ethanol
4. Mix this solution with 100 ml of 85 % phosphoric acid.
5. Make up volume to 1 L with deionized water
6. Filter the solution through Whatman no. 1 filter paper and then store in an
amber bottle at room temperature.
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7. The solution should last for several weeks, however, if you notice precipitate
then refilter before use.
Stock protein Standard:
1. Make a stock solution of Bovine Serum Albumin (BSA) at a concentration of
1mg/mL. This should be stored frozen at -20˚C.
2. Since the moisture content of solid protein may vary during storage, the
precise concentration of protein in the standard solution should be
determined from its absorbance at 280 nm. The absorbance of 1mg/mL
solution of Bovine Serum Albumin is 0.66.
Calibration Curve and assay samples:
1. Pipet volumes of 10, 20, 40, 60, 80, and 100 uL of the stock protein standard
(1mg/mL solution of Bovine Serum Albumin) in to clean glass test tubes.
2. Using distilled water, make each test tube up the voulume of 100 uL.
3. Create a blank with 100 uL of just distilled water.
4. Pipet 100 uL Leaf extract sample in to clean glass test tubes and develop a
renge of delutions (1, 1:10, 1:100, 1:1000).
5. Add 5 mL of the protein reagent to each of the test tubes and mix by gently
vortexing or inversion.
6. Using a spectrophotometer set to A595, measure each of the samples against
the reagent blank between 2 min and 1 hour after mixing.
7. Develop a standard curve to determine the concentration of the unknown
sample.
Personal Protective Equipment / Engineering Controls:
Lab coat, eye protection and hand protection must be selected as required by Section D of the EH&S
Laboratory Safety Manual. Check the box(es) next to the item(s) which are appropriate for your
procedure.
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Ethanol:
Eye Contact Rinse immediately with plenty of water, also under the eyelids, for at least 15 minutes. Obtain
medical attention.
Skin Contact Wash off immediately with plenty of water for at least 15 minutes. Obtain medical attention.
Inhalation Move to fresh air. If breathing is difficult, give oxygen. Do not use mouth-to-mouth resuscitation if
victim ingested or inhaled the substance; induce artificial respiration with a respiratory medical device.
Obtain medical attention.
Ingestion Do not induce vomiting. Obtain medical attention.
Phosphoric acid:
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Eye Contact Rinse immediately with plenty of water, also under the eyelids, for at least 15 minutes.
Immediate medical attention is required.
Skin Contact Wash off immediately with plenty of water for at least 15 minutes. Immediate medical attention
is required.
Inhalation Move to fresh air. If breathing is difficult, give oxygen. Do not use mouth-to-mouth resuscitation
if victim ingested or inhaled the substance; induce artificial respiration with a respiratory
medical device. Immediate medical attention is required.
Ingestion Do not induce vomiting. Call a physician or Poison Control Center immediately
Coomassie blue:
If inhaled
If breathed in, move person into fresh air. If not breathing, give artificial respiration.
In case of skin contact
Wash off with soap and plenty of water.
In case of eye contact
Flush eyes with water as a precaution.
If swallowed
Never give anything by mouth to an unconscious person. Rinse mouth with water.
Bovine Serum Albumin
If inhaled
If breathed in, move person into fresh air. If not breathing, give artificial respiration.
In case of skin contact
Wash off with soap and plenty of water.
In case of eye contact
Flush eyes with water as a precaution.
If swallowed
Never give anything by mouth to an unconscious person. Rinse mouth with water
Handling & Storage Precautions:
Phosphoric acid:
Handling Use only under a chemical fume hood. Wear personal protective equipment. Do not get in
eyes, on skin, or on clothing. Do not breathe vapors/dust. Do not ingest.
Storage Keep containers tightly closed in a dry, cool and well-ventilated place. Corrosives area.
Ethanol:
Handling Wear personal protective equipment. Ensure adequate ventilation. Use explosion-proof
equipment. Keep away from open flames, hot surfaces and sources of ignition. Take
precautionary measures against static discharges. Do not breathe vapors or spray mist. Do not
get in eyes, on skin, or on clothing. Do not ingest.
Storage Keep containers tightly closed in a dry, cool and well-ventilated place. Keep away from open
flames, hot surfaces and sources of ignition. Flammables area.
Coomassie blue:
Precautions for safe handling
Provide appropriate exhaust ventilation at places where dust is formed. Normal measures for preventive fire
protection.
Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place.
Keep in a dry place.
Bovine Serum Albumin:
Precautions for safe handling
Provide appropriate exhaust ventilation at places where dust is formed.
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Conditions for safe storage
Keep container tightly closed in a dry and well-ventilated place.
Recommended storage temperature: 2 - 8 °C
Light sensitive. Keep in a dry place
Waste Disposal Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess
solutions) must be placed in an appropriately labeled hazardous waste container for EH&S
disposal. Compatible substances may be combined into one waste container.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Use personal protective equipment. Ensure adequate ventilation. Remove all sources of
ignition. Take precautionary measures against static discharges. Do not get in eyes, on skin, or on clothing.
Health & Safety Summary for Required Reagents:
In case of Ethanol:
Inhalation
May be harmful if inhaled. May cause respiratory tract irritation.
Skin
May be harmful if absorbed through skin. May cause skin irritation.
Eyes
May cause eye irritation.
Ingestion
May be harmful if swallowed.
In case of Phosphoric Acid:
Inhalation
May be fatal if inhaled. Material is extremely destructive to the tissue of the mucous
membranes and upper respiratory tract.
Skin
Harmful if absorbed through skin. Causes skin burns.
Eyes
Causes eye burns.
Ingestion
Harmful if swallowed.
In case of Coomassie Blue:
Inhalation
May be harmful if inhaled. May cause respiratory tract irritation.
Skin
May be harmful if absorbed through skin. May cause skin irritation.
Eyes
May cause eye irritation.
Ingestion
May be harmful if swallowed.
In case of Bovine Serum Albumin:
Inhalation
May be harmful if inhaled. May cause respiratory tract irritation.
Skin
May be harmful if absorbed through skin. May cause skin irritation.
Eyes
May cause eye irritation.
Ingestion
May be harmful if swallowed.
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Brief description of associated hazards:
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Ethanol
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Target
Organ(s)
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Eyes, Skin,
Reproductive
System, Central
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Incompatibilities
Strong oxidizing
agents, Strong acids,
Acid anhydrides,
Alkali metals,
Ammonia, Peroxides
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3 0 0
Strong acids and
oxidizing agents
Strong acids and
oxidizing agents
0 0 0
 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the
contents of the entire MSDS(s) before starting this procedure.
References:
Walker, John M., The Protein Protocols Handbook. 2nd Edition. Chapter 4. The Bradford
Method for Protein Quantification.
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0 0 0