Clinical Chemistry (CHE 221)

Experiment # 9 Total Protein, Albumin, and the A/G Ratio

Name ______________________________

Date Performed______________________

Date Submitted______________________

Partners Name(s)_____________________

Partners Name(s)_____________________

Experiment title: Total Protein, Albumin, and the A/G Ratio Reading assignment: Bishop 6th edition pages 252-254, and the attached protocols. Pre-lab questions

None

Protocol(s) Part I. Determination of total serum protein by Biuret reagent (protocol attached). Part II. Determination of serum albumin by reaction with bromocresol purple (protocol attached). Part III. Calculation of the Albumin to Globulin (A/G) ratio. Part IV. Determination of the total serum protein by refractometry.

1) Your instructor will demonstrate the use of the refractometer. 2) A series of dilute standards prepared from the Level II Standard as shown in table

1 will be provided. 3) Measure the refractive index of all of the standards provided and one patient

sample. 4) Prepare a calibration curve from the standard data by graphing Refractive Index

versus Total Protein Concentration. In order to do this you will have to calculate the protein concentration of each of the standards for which the refractive index was measured, using the volumes in table 1.

5) Use the calibration curve to determine the patient’s total serum protein concentration.

2000 L total protein reagent

40 L patientserum

40 L water 40 L level Istandard

incubate for five minutesat room temperature

1) zero instrument with blank at 540 nm2) measure absorbance at 540 nm

Total Protein Flowchart

Blank Standard Patient

2000 L albumin reagent

20 L patientserum

20 L water 20 L level Istandard

incubate for one minuteat room temperature

1) zero instrument with blank at 630 nm2) measure absorbance at 630 nm

Albumin Flowchart

Blank Standard Patient

Results

Part I. Determination of Total Protein by Absorbance (Biuret Reagent)

Absorbance Level I Standard at 540 nm_________

Absorbance Patient Sample at 540 nm_________

Calculations – Level I Standard has Total Protein concentration of 4.6 g/dL

Total Protein Concentration_________ g/dL

Part II. Determination of Albumin by Absorbance (bromocresol purple)

Absorbance Level I Standard at 600 nm_________

Absorbance Patient Sample at 600 nm_________

Calculations - Level I Standard has an Albumin concentration of 3.1 g/dL

Albumin Concentration_________ g/dL

Part III. Calculation of the Albumin to Globulin (A/G) Ratio

Calculate the concentration of globulins from the equation

Globulin Concentration = Total Protein – Albumin

Globulin Concentration__________ g/dL

Calculate the Albumin to Globulin ratio

A/G Ratio_________

Part IV. Determination of Total Protein Concentration in Serum by Refractometry

Standard Sample Number

Volume Level II

Standard (L) Volume Water (L)

Total Protein Concentration

ND

1 2000 0

2 1500 500

3 1000 1000

4 500 1500

5 250 1750

6 200 1800

7 150 1850

8 100 1900

9 50 1950

10 0 2000

Patient Sample

- -

Part V. Determination of Total Protein on the Beckman CX3 Run your patient sample on the CX3 to determine the total protein concentration

Post-lab questions

1) Using the total serum protein value you obtained on the CX3 in part V as the accepted value, calculate the percent error in your experimental determination of the patient’s total protein by reaction with Biuret reagent in part I.

2) Using the total serum protein value you obtained on the CX3 in part V as the accepted value, calculate the percent error in your experimental determination of the patient’s total protein by refractometry in part IV.