This is my protocol:
- Pipette 5 mL of milk in centrifuge tubes.
- Add 17 mL of 7.5% trichloroacetic acid (TCA) (w/v).
- Centrifuge at 3000 rpm for 10 min.
- Pipette 2 mL of the supernatant & make up vol to 5 mL with distill water.
- Add 5 mL of 0.02 M TBA using vortex mixer in a screw cap tubes.
- To prepare Blank, add 5 mL of 0.02 M TBA & 17 mL of 7.5% TCA to 5 mL DW in screw cap tubes & Label Blank.
- Heat mixture for 40 min at 100oC. Slightly loose the cap to prevent pressure build up.
- Allow samples to cool at room temperature.
- Pipette 200 µL of each sample into microplate in triplicate.
- Using microplate reader, read the absorbance at 532 nm.
- Prepare standard curve using tetraethoxypropane (TEP) soln & expressed as mg MDA/L.
calculation mg/l: absorbance of sample*calibration factor*final vol (ml)/ sample vol(ml)
calibration factor = 1/ slope of calibration graph
slope= dY/dX
where Y= absorbance and X= concentration of standard in ppm,
plot calibration graph of concentration (on X axis) Vs Absorbance on (Y axis)
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