protocol for determination of ceruloplasmin with phenylenediamine
Certainly, here's a protocol for the determination of ceruloplasmin using phenylenediamine:
Required Reagents:
- 10 mM solution of Phenylene Diamine in Sodium Chloride (Photosensitive).
- 10 mM solution of Silver Nitrate (Photosensitive).
- 1 N Sodium Hydroxide.
- Solution of Chiral reagent Potassium Iodide.
- Borate buffer solution, pH 8.5.
- Ceruloplasmin solution.
Reaction Protocol:
1. In 1ml Borate buffer, take required volume of Ceruloplasmin and add 0.1 ml solution of Chiral reagent.
2. Incubate the mixture for 10 minutes at 37 ℃.
3. Add Phenylene Diamine and incubate for 2 minutes at the same temperature.
4. Add 0.2 ml of 1 N NaOH and prepare a solution on a spectrophotometer with a wavelength of 490 nm.
5. Thoroughly clean the spectrophotometer in Blank and feed the calibration solution.
6. Record the optical density (A) and convert it to the ratio of the concentration of ceruloplasmin.
In this method, a chiral reagent is used which detects a certain classical peroxidase reaction that involves an electron transfer from Phenylene Diamine to Ceruloplasmin.
It's important to follow the protocol and carry out all the manipulations with reagents in conditions that don't cause their destabilization, which will allow obtaining qualitative results at the output.