Lab 2

HUMAN BIOCHEMISTRY HDB10803
UNIVERSITI KUALA LUMPUR

INSTITUTE OF MEDICAL SCIENCE TECHNOLOGY
LABORATORY REPORT
SUBMISSION FORM
To: MRS.NURSHUKRIYAH BT MOHAMZAD Code Subject: HDD10803 HUMAN
BIOCHEMISTRY
NAZIR
From: LUQMAN AL HAKIM BIN MIOR SHAHRIMAN Student ID. No.: 12105122086
MITHESH KUMAR A/L P.R. SURESH KUMAR 12105022024
MUHAMMAD HAFIZI BIN ABDUL AZIZ 12105122040
AMMAR ZAIMI BIN AZRUELL AZLA 12105322028
UTHMAN AL AFFAN BIN ALIAS 12105322029
AMIER HAFIZIE BIN SUKHAIMI 12105322010
Title of Experiment: DETERMINATION OF PROTEIN IN AN UNKNOWN SERUM
Note: Submission is after 1 week. Late submission will be penalized.

*To be filled by the marker*
VERY POOR GOOD VERY EXCELLENT
CRITERIA POOR GOOD
1 2 3 4 5
1.0 INTRODUCTION & OBJECTIVES
1. State the background to the experiment
3 6 9 12 15
conducted.
2. State the objectives of the experiment
2.0 METHODOLOGY
1. Summarize step by step used in performing this 3 6 9 12 15
experiment.
3.0 RESULTS AND DISCUSSIONS
1. Data are presented as deemed suitable with
complete label and units in tables and/or graphs.
2. Explanations of the referred tables and/or graphs 8 16 24 32 40
are presented after it.
3. Discuss on the findings and relations to the
theory and objective of experiment.
4.0 CONCLUSIONS
1. Summary of the results to relate the findings or 3 6 9 12 15
results with the theory applicable to the experiment.
5.0 REFERENCES 1 2 3 4 5
6.0 FORMAT AND LANGUAGE 2 4 6 8 10
TOTAL
1
Date of Submitted:
Student Name:
Student ID:
Subject/Code:
Experiment title:
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Lecture Name: Stamp/Signature
Student Slip Received

Date of Submitted:
Student Name:
Student ID:
Experiment title:
Office Slip
2
SAMPLE
UNIVERSITI KUALA LUMPUR
DETERMINATION OF PROTEIN IN AN UNKNOWN SERUM
Introduction:
Protein is a macromolecule that constructed from one or more unbranched chains of

amino acid. Protein is polymer of amino acid and the structure of Amino acid consist amino
group and carboxyl group and R- group . In this world there are twenty common of amino
acid and in this experiment we aim to determine protein using unknown serum.
Learning Objectives:
1. To be able to prepare a Bradford reagent for protein assay.

2. To understand the concept of serial dilutions and its purpose.
3.To construct a standard curve for protein level determination.
4.Perform quantification of protein in an unknown solution.
Equipment and Materials:
Spectrophotometer,
Cuvettes, 10ml volumetric flask,
10, 50 ml beaker,
Biuret Reagent.
Calibrated pipette (0-50 µL, 1ml) and tips (blue and yellow),
Test tubes,
Bovine Serum Albumin - BSA,
Unknown serum.
Methods:
Part A: Preparation of Bradford Reagent
1. Dissolve 2.5 mg of Coomassie Brilliant Blue G-250 in 2.5 ml of Ethanol.

2. Add 10 ml 85% (w/v) phosphoric acid (H3PO4).
3. Add the acid solution mixture slowly into 85 ml of H2O and let the dye dissolve
completely.
4. Store in a dark bottle at 4°C.
5. Filter using Whatman #1 paper to remove the precipitates just before use.
Part B: Serial Dilution & Measurement of Unknown
3
1. Prepare five standard solutions (2ml each) containing 0, 10, 20, 30, 40, and 50 µg/ml
BSA.
2. Protein solutions are normally assayed in duplicate or triplicate.
3. Pipet 800 µl of each standard (6 std X duplicate) into clean, dry test tube.
4. For blank, 800 µl H20 into new test tube (duplicate).
5. For the unknown sample, add 800 µl of serum into new test tube (duplicate).
6. Add 200 µl of dye reagent concentrate to each tube and vortex.
7. Incubate at room temperature (RT) for at least 5 min.
8. Absorbance will increase over time; samples should incubate at RT for no more than
1 h.
9. Measure absorbance at 500 nm.
Safety and precaution (if any):
To follow the laboratory Safety rules.
Result:
4
No Protein concentration (µg/mL) Absorbance

1. 0 0
2. 10 0.20
3. 20 0.25
4. 30 0.38
5. 40 0.50
6. 50 0.62
7. Unknown 0.40
Conclusion:
To conclude, by using this method, we can determine concentration level of protein serum.
The experiment has proven the hypothesis, that higher protein concentration will increase
the net absorbance.
References:
1.Bradford, MM. (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72,
248-254.
2’Smith, P.K, Krohn R.I., Hermanson G.T., et al. (1987) Measurement of protein using
bicinchoninic acid. Analytical Biochemistry. 150, 76-85.
3.Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, Current
Protocols in Cell Biology, A.3H.1-A.3H.28, John Wiley & Sons, Inc.

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HUMAN BIOCHEMISTRY HDB10803

UNIVERSITI KUALA LUMPUR

Title of Experiment: DETERMINATION OF PROTEIN IN AN UNKNOWN SERUM

Note: Submission is after 1 week. Late submission will be penalized.

Student Slip Received

Protein is a macromolecule that constructed from one or more unbranched chains of

1. To be able to prepare a Bradford reagent for protein assay.

Equipment and Materials:

1. Dissolve 2.5 mg of Coomassie Brilliant Blue G-250 in 2.5 ml of Ethanol.

Part B: Serial Dilution & Measurement of Unknown

Safety and precaution (if any):

To follow the laboratory Safety rules.

No Protein concentration (µg/mL) Absorbance

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