6. Quantitative determination of rat peroxisome proliferators activators γ (PPAR-γ):
Rat PPAR-γ concentration was determined by ELISA; using a commercially available kit, MyBioSource, Inc., California, USA. The kit utilizing a monoclonal anti- PPAR-γ antibody and an PPAR-γ-HRP conjugate enzyme-linked immunosorbent process assay (ELISA) to determine the concentration of PPAR-γ in serum, plasma, urine, cell culture supernatant and tissue homogenates.
Principle of the assay:
The sample and buffer have been pre-coated with PPAR-γ-HRP conjugate. then incubation is carried out, followed by washing to remove the uncombined enzyme. Upon adding substrate for HRP enzyme, the color of the liquid will change into blue. Finally, a stop solution is added to stop the reaction, …show more content…
The desired number of coated wells was secured in the holder.
2. Blank wells, standard wells, and test sample wells were set respectively:
• Blank well: 100 μl of PBS was added.
• Standard wells: 100 μl of each standard was added to each corresponding standard well.
• Test sample wells: 100 μl of sample was added
10 uL of Balance Solution was Dispensed into 100 uL specimens.
50 μl of conjugate was added into each well.
3. The plate was sealed, and gently shaken, then incubated 60 minutes at 37 ℃.
4. The plate was washed using Manual Washing; the plate was emptied by inverting it and shaking the content out, and tapped on absorbent papers to dry.
5. 350 μl washing solution was added into each well, and left for 1-2 minutes. This process was repeated five times.
6. Fifty μl of substrate solution A was added, to each well, and then 50 μl of substrate solution B was added to each well.
7. The plate was gently shaken and incubated for 15 minutes at 37℃ away from light.
8. Fifty μl Stop Solution was added into each well to stop the reaction.
9. The absorbance was measured at 450 nm wavelength within 15 minutes after adding the stop solution using a microtiter plate reader.
Rat PPAR-γ concentration was determined by ELISA; using a commercially available kit, MyBioSource, Inc., California, USA. The kit utilizing a monoclonal anti- PPAR-γ antibody and an PPAR-γ-HRP conjugate enzyme-linked immunosorbent process assay (ELISA) to determine the concentration of PPAR-γ in serum, plasma, urine, cell culture supernatant and tissue homogenates.
Principle of the assay:
The sample and buffer have been pre-coated with PPAR-γ-HRP conjugate. then incubation is carried out, followed by washing to remove the uncombined enzyme. Upon adding substrate for HRP enzyme, the color of the liquid will change into blue. Finally, a stop solution is added to stop the reaction, …show more content…
The desired number of coated wells was secured in the holder.
2. Blank wells, standard wells, and test sample wells were set respectively:
• Blank well: 100 μl of PBS was added.
• Standard wells: 100 μl of each standard was added to each corresponding standard well.
• Test sample wells: 100 μl of sample was added
10 uL of Balance Solution was Dispensed into 100 uL specimens.
50 μl of conjugate was added into each well.
3. The plate was sealed, and gently shaken, then incubated 60 minutes at 37 ℃.
4. The plate was washed using Manual Washing; the plate was emptied by inverting it and shaking the content out, and tapped on absorbent papers to dry.
5. 350 μl washing solution was added into each well, and left for 1-2 minutes. This process was repeated five times.
6. Fifty μl of substrate solution A was added, to each well, and then 50 μl of substrate solution B was added to each well.
7. The plate was gently shaken and incubated for 15 minutes at 37℃ away from light.
8. Fifty μl Stop Solution was added into each well to stop the reaction.
9. The absorbance was measured at 450 nm wavelength within 15 minutes after adding the stop solution using a microtiter plate reader.