OHS026
Safe Work Procedure
Faculty/Division:
Medicine
School/ Divisional Unit
School of Medical Sciences ORU/NRM
Document number
SOMS.CGM.SWP002
Initial Issue date
26-6-09
Current version
1.0
Current Version
Issue date
26-6-09
Next review date
26-6-12
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
form.
Safe Work Procedure Title and basic description
Title: BCA Protein Assay
Description: Quantifies amount of protein in samples of lysed cell extracts
Associated risk assessment title and location: 02_RA_BCA Protein Assay_JC
Describe the activity or process
The BCA Protein Assay is a complete kit supplied by Thermo Scientific (formerly Pierce). The procedure is as per the instructions
supplied by the manufacturer. The assay can be performed in test-tube volumes to be read via cuvette in a spectrophotometer, or
scaled-down to microplate volumes to be read on a plate reader.
Preparation of Standards and Working Reagent (required for both assay procedures)
Preparation of Diluted Albumin (BSA) Standards
Use Table 1 as a guide to prepare a set of protein standards. Dilute the contents of one Albumin Standard (BSA)
ampule into several clean vials, preferably using the same diluent as the sample(s). Each 1 ml ampule of 2.0 mg/ml
Albumin Standard is sufficient to prepare a set of diluted standards for the working range suggested in Table 1. There
will be sufficient volume for three replications of each diluted standard.
Table 1. Preparation of Diluted Albumin (BSA) Standards
Dilution Scheme for Standard Test Tube Protocol and Microplate Procedure (Working Range = 20–2,000 μg/ml)
Vial
Volume of Diluent
Volume and Source of BSA
Final BSA Concentration
A
0
300 μl of Stock
2,000 μg/ml
B
125 μl
375 μl of Stock
1,500 μg/ml
C
325 μl
325 μl of Stock
1,000 μg/ml
D
175 μl
175 μl of vial B dilution
750 μg/ml
E
325 μl
325 μl of vial C dilution
500 μg/ml
F
325 μl
325 μl of vial E dilution
250 μg/ml
G
325 μl
325 μl of vial F dilution
125 μg/ml
H
400 μl
100 μl of vial G dilution
25 μg/ml
I
400 μl
0
0 μg/ml = Blank
Test-tube Procedure (Sample to Working Reagent (WR) ratio = 1:20)
1. Pipette 0.1 ml of each standard and unknown sample replicate into an appropriately labelled test tube.
2. Add 2.0 ml of the WR to each tube and mix well.
3. Cover and incubate tubes at selected temperature and time:
Standard Protocol: 37°C for 30 minutes (working range = 20-2,000 μg/ml)
RT Protocol: RT for 2 hours (working range = 20-2,000 μg/ml)
NB: Increasing the incubation time or temperature increases the net 562 nm absorbance for each test and decreases
both the minimum detection level of the reagent and the working range of the protocol. Using a forced-air incubator
can introduce significant error in colour development because of uneven heat transfer.
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
4. Cool all tubes to RT.
5. With the spectrophotometer set to 562 nm, zero the instrument on a cuvette filled only with blank from standard
curve. Subsequently, measure the absorbance of all the samples within 10 minutes.
6. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 nm absorbance
measurement of all other individual standard and unknown sample replicates.
7. Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard vs.
its concentration in μg/ml. Use the standard curve to determine the protein concentration of each unknown sample.
Note: If sample size is limited, 10 μl of each unknown sample and standard can be used (sample to WR ratio = 1:20).
However, the working range of the assay in this case will be limited to 125-2,000 μg/ml.
Microplate Procedure (Sample to WR ratio = 1:20~1:8)
1. Pipette 10-25 μl of each standard or unknown sample replicate into a microplate well (working range = 20-2,000
μg/ml). It will probably be necessary to dilute your samples in order to fit into the detectable range of the assay. For
tissue culture samples this dilution may be between 1:5 – 1-20; for organ tissue samples higher dilutions such as 1:50
may be necessary. This dilution factor will have to be accounted for when inferring concentrations from your standard
curve.
2. Add 200 μl of the WR to each well and mix by gently shaking.
3. Cover plate and incubate at 37°C for 30 minutes. Incubation at RT for 1 hr also works well.
4. Cool plate to RT.
5. Measure the absorbance at or near 562 nm on a plate reader.
Notes: Wavelengths from 540-590 nm have been used successfully with this method.
Because plate readers use a shorter light path length than cuvette spectrophotometers, the Microplate Procedure
requires a greater sample to WR ratio to obtain the same sensitivity as the standard Test Tube Procedure.
If higher 562 nm measurements are desired, increase the incubation time to 2 hours.
Increasing the incubation time or ratio of sample volume to WR increases the net 562 nm measurement for each well
and lowers both the minimum detection level of the reagent and the working range of the assay. As long as all
standards and unknowns are treated identically, such modifications may be useful.
6. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 nm
measurements of all other individual standard and unknown sample replicates.
7. Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard vs.
its concentration in μg/ml. Use the standard curve to determine the protein concentration of each unknown sample.
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc





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BCA Protein Assay Kit (Pierce)
Microplate reader
Disposable Face Mask
Gloves
Safety Glasses
Lab Coat/Gown
List potential hazards and risk controls including
specific precautions required


BCA protein assay kit: Reagents A and B can cause irritation by eye and skin contact, and
inhalation. Wear gloves and gown and safety glasses when handling the kit. Must read MSDS
FOR KITS before the start of the experiment.
Eye wash and shower station if contact occurs
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List emergency shutdown instructions
In the event of inhalation or ingestion the first aid officer should be consulted immediately.
First Aid Officer: Renee Szokolai ext 58497
List clean up and waste disposal requirements
Plastic tubes and plates should be disposed in correct biological waste bags.
List legislation, standards and codes of practice
used in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3: 2006 Safety in laboratories Part 3: Microbiological aspects and containment facilities
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Supervisory approval, training, and review
Supervisor: Peter Gunning
Signature:
Plant custodian: n/a
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction to Lab, Training in this SWP
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Page 3 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007