Addis Ababa University

College of Health Sciences

School of Medicine

Department of Biochemistry

Laboratory Report for

TOTAL PROTEIN

Sintayehu Melese
UGR/7401/13
Mar-6-2022
 Total protein test
Objective

To measure the total amount of protein (albumin and globulin) in the body in order to determine
whether the levels of protein in your body are adequate or not.

Principle

Polypeptides containing at least two peptide bonds react with biuret reagent. In alkaline solution,
proteins bind with the cupric ions present in the biuret reagent to form a blue-violet colored
complex. The cupric ion forms a coordination complex with protein nitrogen with very little
difference between albumin and globulin on a protein-nitrogen basis. The intensity of the color
formed is directly proportional to the amount of proteins present in the sample.

Materials used
 Test tube
 Test tube rack
 Micropipettes
 Micropipette tips
 Incubator
 Spectrophotometer capable of reading at 540nm
 Stopwatch
 Water bath at 37 degree centigrade
 Cuvettes with light path of 1cm

Chemicals Used

R1: Biuret reagent

CAL: Protein standard (6 g/dl)

Procedure

1. Take 3 clean and dry test tubes and label them B(Blank), T(standard), S(sample)
respectively.
2. Add 1 ml of biuret reagent (R1) in each of the test tubes.
3. Add 20µl of sample serum in the ‘S’ labeled test tube, 20µl of standard protein in
the ‘T’ labled test tube and 20µl of distilled water in ‘B’ labeled test tube.
4. Shake well and allow the mixtures to stand in the water bath for 10 minutes.
5. Observe for any color change.
6. After 10 minutes transfer the mixtures in the test tube in to 3 separate cuvettes.
7. Put the blank specimen in to the spectrometer of 540nm for calibration.
 8. Put the rest cuvettes into cuvette holder in the spectrometer one after the next and
record the results of each.

BLank Sample CAL(standard)

R1 1.0 ml 1.0 ml 1.0 ml
Sample - 20µl -
CAL(standard) - - 20µl

Calculation:
Absorbance of test ( AT )
Sample test concentration = Absorbance of standard ( AS) ×6 g /dl

Result:
Absorbance Transmission
blank 0 100%
sample 0.581 26.2%
CAL 0.395 40.3%

From this table the sample test protein concentration will be 8.82 g/dl.

Reference value:
Adults 6.6-8.7 g/dl
Premature 3.6-3.6 g/dl
Newborn 5.3-8.9 g/dl
Pregnant Concentration lowers from 6.9 to 6.1 g/dl

Discussion:

The amount of serum total protein to be normally found is up to 6 - 8.7 g/dl in

adults when the temperature is 37°c. Taking this into consideration, the amount of
total protein in the sampled serum was 8.82 g/dl and was only slightly deviated
from the normal range. Though it may show that there is some problem in
measurements and calibration, it was reported that such levels are still close
enough to be accounted normal in an adult.

Limitation
The spectrometer with a thermostat might have caused the rough surfaces of the cuvettes to get
wet and absorb some light while incubating.
There were some technical errors while sampling the serum, and the photometer was
reported to not be calibrated well enough to give adequate results.
Reference:
David L. Nelson, Micheal M. Cox. Lehninger: Principles of Biochemistry. 5th edition. New York,
NY: W.H. Freeman and Company