THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY

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1 Page: 1 of 4 1. Risk Assessment: This risk assessment has been prepared using the National Safety Council of Australia Risk Score Calculator and risks have been determined to be as stated. 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 equipment. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. Micro-measurements of Protein levels. TASK PERFORMED HAZARDS 1. Reagents: Folin Ciacolteu Reagent is Corrosive RISK ASSESSMENT 1. The risk from this reagent is low if appropriate PPE is followed. RISK CONTROL 1. Wear safety glasses, laboratory gown and gloves when handling this reagent and solutions containing this reagent. Avoid breathing vapours. 2. Purpose: 2.1 To accurately measure the levels of protein in small quantities of samples, comparing to a standard curve. 3. Equipment: 3.1 Pipettors 3.2 Spectrophotometer 3.3 Vortex WRITTEN BY NAME (signed) Lynn Herd DATE 23 rd June 2003 CHECKED BY Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 4 4. Reagents: 4.1 2% Na 2 CO 3 in 0.1M NaOH 4 g Sodium Carbonate 0.8 g Sodium Hydroxide Make up to 200 mls with dd H 2 O % NaK Tartrate 1 g Sodium Potassium Tartrate Make up to 50 mls with dd H 2 O % CuSO g Copper Sulphate Make up to 20 mls with dd H 2 O mg/ml BSA 0.05 g Bovine Serum Albumin Make up to 50 mls with dd H 2 O or 0.1 M NaOH. Aliquot into 500 ul amounts in microfuge tubes and store frozen at 20 o C until required. 4.5 Reagent A ml 2% Na 2 CO 3 in 0.1M NaOH 750 ml 2% NaK Tartrate 750 ml 1% CuSO 4 Make up fresh before use. 4.6 Reagent B 1.5 ml Folin Ciocalteu Reagent see NOTE 1.5 ml dd H 2 O Make up fresh before use. (****NOTE**** Folin Ciocalteu Reagent HAZARD - Corrosive: Wear safety glasses, gloves and laboratory gown when handling. Causes burns. Danger of cumulative effects. Risk of serious damage to eyes. Do not breathe gas/fumes/vapour/spray.

3 Page: 3 of 4 5. Procedure: 5.1 Set up standard curve. In disposable 3DT tubes dilute the BSA Standard as follows (each standard should be set up in triplicate): BSA Standard (1mg/ml) Distilled H 2 O Protein Concentration Standard 1-60 ul 0 Standard 2 15 ul 45 ul 0.25 mg/ml Standard 3 30 ul 30 ul 0.50 mg /ml Standard 4 45 ul 15 ul 0.75 mg /ml Standard 5 60 ul mg /ml 5.2 Set up samples to be assayed (in triplicate if possible) Dilute samples as required to bring protein concentration within range of standard curve If dealing with an unknown protein concentration, prepare serial dilutions 1, 1/10, 1/100, 1/1000 and assay all Pipette 60 ul of sample / dilution into 3DT tube. 5.3 Add 1 ml Reagent A to all tubes and vortex. Leave 10 minutes at room temperature. 5.4 Add 100 ul Reagent B to all tubes and vortex. Allow to stand at room temperature for 30 minutes. 5.5 Read optical density (Absorbance) in the spectrophotometer at 550 nm using 1 ml cuvettes or the automated cell. Zero the spectrophotometer on Standard 1 (0 mg/ml protein), then read the other standards. 5.6 Use the Standard Curve option on the Spectrophotometer to draw up the standard curve. Check the % CV and R-squared value as a control for the Standard Curve. 5.7 After accepting the standard curve, read the samples. The dilution factor may be entered into the Spectrophotometer for automatic calculation of the Protein level of the original sample. 5.8 In a range of dilutions, the sample value considered to be most accurate will be the one whose absorbance reading lies closest to the middle of the standard curve absorbance readings.

4 Page: 4 of 4 6. Safety Precautions: 6.1 Good laboratory techniques are to be used at all times. 7. Shutdown: 7.1 Lowry protein waste solutions may be disposed of down the sinks. Run the tap water afterwards to dilute further. 7.2 Tubes should be disposed of as contaminated waste. 7.3 Check that the Visible Light has been turned OFF on the Spectrophotomoter. 7.4 Rinse the automated cell on the Spectrophotometer with water. 8. Change History: 8.1 Issue Number: 1st Issue Date Issued: 8.2 Issue Number: Date Issued: Reason for Change:

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