clinical chemistry (che 221) experiment # 9 total protein
TRANSCRIPT
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.