THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 052 MOD: 1st Issue Page: 1 of 5

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1 Page: 1 of 5 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED The measurement of protein levels using a very small volume of sample. HAZARDS 1. Chemical Hazard BCA Protein Assay Reagent Kit (Harmful) - BCA Protein Assay Reagent B (Toxic) R.S. 942 RISK ASSESSMENT 1. The risk from chemical contact is low if risk controls are followed. RISK CONTROL 1. Wear lab coat, enclosed shoes and safety glasses when handling BCA Protein Reagents. 2. Training should be undertaken in General Laboratory Safety. 3. Consult the GEP 063 for the Risk Assessment and Instructions for operating the Fluorescent Plate Reader. 2. Purpose: 2.1. To measure the protein levels in given samples, referring to a standard curve. The sample volumes used are very small, and the assay can be read using a microplate reader which records and calculates results using the accompanying standard curve. WRITTEN BY MODIFIED BY AUTHORIZED BY NAME (signed) Lynn Herd Nikki Verrils Phil Dickson DATE 9 th November th November th November 2004 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 5 3. Equipment: 3.1. Microplate Reader FLUOstar Fluorescent Microplate Reader located in LS3-26 or BioRad Microplate Reader, located in LS3-27 (Cancer Research) Incubator 37ºC 3.3. Pipettors 4. Materials: well microplate flat bottomed, clear 4.2. Pipette tips 4.3. Pierce BCA Protein Assay Reagent Kit (#23225) from Progen 5. Reagents: 5.1. BCA Reagent Mix 19.6 ml BCA Reagent A 0.4 ml BCA Reagent B Mix together when required for procedure. 200ul is added per well, so a smaller volume of Reagent Mix should be prepared as required. Reagent Mix is stable for several days when stored in a closed container at room temperature Albumin Standard Ampoules 2 mg/ml 10 x 1ml ampoules provided with the kit Protein Standard 2 mg/ml 80 mg Bovine Serum Albumin Make up in 40 ml dd H 2 O, aliquot in 1ml lots in microfuge tubes and store at -20ºC until required. 6. Set Up: 6.1. Ensure that you have read and understood the Safety Precautions (Section 6 of this SOP) prior to commencing this procedure 7. Safety Precautions: 7.1. Good laboratory techniques are to be used at all times.

3 Page: 3 of 5 8. Method: 8.1. Assay is usually performed in triplicate and the average value used for calculations Microplate set-up The microplate should be set-up or organized in such a manner that the replicate standards and samples are together. eg Standard Curve Preparation: Prepare the following solutions to be used to run the standard curve. Standard Standard - 2mg/ml (ul) dd H 2 O (ul) or Buffer Protein Concentration (mg/ml) ul x Std Add 25 ul of each standard to its respective well of the microplate.

4 Page: 4 of Samples may either be diluted prior to protein estimation, or sample volumes ranging from 0.5 to 25ul may be added to the respective well of the microplate. Remember to add the equivalent volume of dd H 2 O or Protein Buffer to bring the total well volume to 25ul. Ideally samples should be assayed in triplicate for greater accuracy Add 200µl BCA Reagent Mix (Reagent 5.1) to each well. Mix Incubate plate for 30 minutes at 37ºC. (Incubator available in LS3.23) If higher measurements are desired, increase the incubation time to 2 hours Cool plate to room temperature The plate may be read on a plate reader with filter in the range of 562nm. The FLUOstar fluorescent microplate reader is set up to read at 492nm, which is not ideal, but OK. It can plot a graph of the standards and then calculate controls and unknowns. Alternatively there is a BioRAD microplate reader available in Cancer Research with a suitable filter of 570nm, which will give raw data from which the user can perform their own calculations. 9. Maintenance: 9.1. Store kit at room temperature. 10. Shutdown: Turn off the FLUOstar or BioRAD microplate reader after use Excess reagent should be collected and disposed of as hazardous waste Dispose of microplate in contaminated waste. 11. References: Instructions, BCA Protein Assay Kit Laboratory Method Molecular Biology, BCA Protein EST Assay Kathy Bradley. 12. Attachments: Instructions, BCA Protein Assay Kit

5 Page: 5 of Change History: Issue Number: 1st Issue Date Issued: 16/11/ Issue Number: Date Issued: Reason for Change: