Make fresh on day of assay in a new Corning tube. Standards. Ferrous sulphate. Prepare 1mM solution: 0.278g FeSO4.7H2O in 1 litre distilled water. Dilute as ...
School of Life Sciences
S.O.P.11 FRAP assay on plasma & faecal extracts
STANDARD OPERATING PROCEDURE FOR FRAP ASSAY ON PLASMA & FAECAL EXTRACTS This is the Standard Operating Procedure for conducting the FRAP assay on plasma & faecal extracts in the School of Life Sciences, The Robert Gordon University, St Andrews Street, Aberdeen. A) Safety considerations 1. read & sign relevant Risk Assessment and S.O.P.s for each item of equipment required 2. goggles, lab coat and surgical gloves used throughout 3. 1% biocide made up to wipe all worksurfaces after procedure 4. ensure that there are no leakage’s in the centrifuge buckets - if so clean with biocide 5. dispose of all disposable materials (tissues, tubes, pipettes etc.) into a Biohazard bag for autoclaving Hazards associated with this procedure Electrical shock, biological contamination, chemical contamination Risks associated with this procedure · Chemical contamination of skin, eyes, clothing · Biological contamination of skin, eyes, clothing · Cuts from damaged glassware Persons at risk when using this procedure Students, Staff, Technicians B) General considerations 1. ensure that technical staff are informed beforehand that there will be material for autoclaving 2. keep all samples/standards on ice when not handling them 3. soak contaminated glassware in Tepol solution prior to washing.
C) Equipment and chemicals required Equipment centrifuge, T529 rotor balance (4 d.p.) Sterile graduated pipettes and bulb 50ml Corning tubes Gilson pipettes and tips Ependorfs UV/vis spectrophotometer Water bath (37oC) with pump to circulate water through spectrophotometer Water bath (37 & 50oC) to store FRAP solutions Disposable plastic cuvettes (Fisherbrand, semi-micro cuvette, 1.6ml, FB55147) Timer Chemicals Acetate buffer: 300mM, pH 3.6 3.1g sodium acetate.3H2O 16ml glacial acetic acid Distilled water to 1 litre Check pH, Store @ 4oC Dilute HCl:
40mM 1.46ml conc. HCl (11M) Distilled water to 1 litre Store @ room temperature
TPTZ (2,4,6-tri[2-pyridyl]-s-triazine): 10mM 0.031g TPTZ in 10ml of 40mM HCl, dissolve @ 50oC in water bath Make fresh on day of assay in a new Corning tube. Ferric chloride: 20mM 0.054g FeCl3.6H2O Dissolve in 10ml distilled water Make fresh on day of assay in a new Corning tube. Standards Ferrous sulphate Prepare 1mM solution: 0.278g FeSO4.7H2O in 1 litre distilled water Dilute as follows to make a series of standards: Standard concentration (mM) 0.1 0.2 0.4 0.6 0.8 1.0
FeSO4.7H2O Solution (ml) 1 2 4 6 8 10
Freeze @-20oC in 0.2ml aliquots in ependorfs
Distilled water (ml) 9 8 6 4 2 0
D) FRAP method (Adapted from Benzie & Strain, 1996, Analytical Biochemistry, 239, 70-76) 1.
Switch water baths & spectrophotometer on
2.
Defrost samples & standards
3.
Prepare FRAP reagent: 200ml acetate buffer 20ml TPTZ solution 20ml FeCl3 solution 24ml distilled water Keep in a dedicated plastic bottle. The solution should be straw coloured, if it is tinged with blue discard and prepare fresh solution after rinsing bottle thoroughly with demonised water. Mix; keep in water bath @ 37oC.
4.
Run a set of blanks first. Place 7 cuvettes in the spectrophotometer. Add 30ul distilled water to each cuvette (let the water run down the side of the cuvette). Add 1ml of FRAP reagent vigorously to each cuvette (using a Gilson pipette) so that the contents mix thoroughly (there is no need to mix the cuvettes further).
5.
Set the spectrophotometer at 593nm, the temperature strip should read 37oC. Set timer for 4 minutes.
6.
After 4 minutes, zero the blank and press run to record the absorbance of the samples. Take a written record of the absorbances then reset the spectrophotometer and prepare the next set of samples.
7.
Discard recorded standards/samples down drain with plenty of water.
8.
Repeat 4-6 with 3 sets of standards, the first of which should always be a blank.
9.
Then repeat 4-6 with samples, always using a blank in cuvette 1. All samples must be run at least in triplicate.
Analysis: Subtract the water blank from the sample/standard values. Construct a linear regression for the standards (absorbance against concentration). Use the regression equation to calculate the FRAP values (units, mM Fe(II) per litre) of the samples.
Amendments to basic FRAP assay for use on aqueous faecal extracts. Two problems occur when analysing faecal extracts in the FRAP assay: 1.
Occurrence of precipitate when mixing extract with FRAP solution. To remove sediment from extract: Defrost extract (takes approximately 1 hr). Transfer 500ml of extract into an ependorf. Centrifuge at 14000g, 4oC for 5 min.
2.
Absorbance values of neat faecal extract are greater than the range of standards and the range of greatest accuracy of the spectrophotometer:
Pipette 100ml of supernatant into a new ependorf. Add 900ml of distilled water to effect a 10-fold dilution. Analyse these diluted supernatants for antioxidant capacity using FRAP assay.
