VLD 421: Veterinary Clinical Biochemistry and Laboratory Diagnosis-II (0+1)

Estimation of Protein

METHOD OF ESTIMATION OF PROTEIN

Principle:

          Proteins in serum react with copper of Biuret Reagent in alkaline medium to form a blue purple complex with absorption maximum at 550 nm.

Sample: Serum or plasma.

Reagents:

• Total Protein Reagent (Biuret Reagent – Potassium iodide, Potassium sodium tartarate, Copper sulphate, NaOH)
• Total Protein Standard (6g%)

Procedure:

 Mark three test tubes as Blank, Standard and Test and proceed as follows:

S.No.	Reagents	Blank (B)	Standard (S)	Test (T)
1.	Biuret Reagent	1.0 ml	1.0 ml	1.0 ml
2.	Total Protein Standard	-	20 m l	-
3.	Serum	-	-	20 m l
4.	Distilled Water	2.0 ml	2.0 ml	2.0 ml

• Mix well and incubate at 37⁰C for 10 minutes.
• Measure the O.D of all the Standard and Test against Blank at 550 nm or using an yellow green filter.

Calculations :

Total Proteins (g/dL) (X) = OD of (T)

                                                ------------- x 6

                                                OD of (S)

Globulins (g/dL)  = (Total protein ) – (Albumin)

 

A/G Ratio =                       (Albumin)

                            -----------------------------

                               (Total Protein) – (Albumin)

Method of Estimation : Biuret Method