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LAB MANUAL
School of Biotechnology
International University VNU-HCMC
CONTENT
PART I. PRACTICAL EXPERIMENTS
EXPERIMENT 1:
Protein quantification applying Hartree-Lowry assay
EXPERIMENT 2:
Exploring the enzymatic activity of bromelain
EXPERIMENT 3:
Soluble carbohydrate quantification applying Anthrone assay
EXPERIMENT 4:
Quantitative determination of calcium in powdered milk
EXPERIMENT 1
Protein quantification applying Hartree-Lowry assay
1. Principle
Protein quantification is highly essential in biochemical research. Several assays
have been developed; however, each has limitation due to biochemical separation
process and purposes of experimenters. Basing on differences in amino acid
content of distinct proteins, currently experimental condition, experiences of
experimenters, protein of interest and their amounts, suitable assays will be
applied to obtain the best outcome with acceptable error. Before deciding which
assay will be used, considering its sensitivity, accuracy, interfering substances and
available timing are highly recommended.
The table below shows you some widely used assays of protein quantification.
Assay
Bradford
Sensitivity
150-750 g/mL
HartreeLowry
30-150 g/mL
Mechanism
Based upon reversible, pH
dependent
binding
of
coomassie brilliant blue G250
dye
to
protein.
Absorbance at 595 nm
Combination of copper and
phosphomolybdic/phospho
tungtic
acid
reacts
quantitatively with protein.
Absorbance at 750 nm
Advantage
Moderately
sensitive,
easy and fast
to run
Disadvantage
Alkaline
pH/buffer will
interfere
Good
sensitivity
Laborious.
Detergent and
chelating agent
can interfere.
Based
on
fluorescence
properties of aromatic amino
acid residue in the protein.
Very
sensitive, fast
and
nondestructive to
sample
Same
concentration
of
different
proteins
can
lead
to
variation
in
reading result
due to various
content
of
aromatic
residue.
Biuret
Fluorescence
emission
5-50 g/mL
Chemicals
3. Practical procedures
3.1 Preparation of sample
Step 1: Get soybeans and pulverize them by blender or stone mortar.
Step 2: Take an exact amount of 5g pulverized soybeans and put it into a stone
mortar.
3.2 Extraction:
1st time:
Step 1: Get about 40mL of distilled water and put small amount of water into
the stone mortar and grind down the sample.
Step 2: Then put the rest of water to the stone mortar and grind down the
sample carefully.
Step 3: Put the extract solution into a beaker. The grounds of the soybean is
still kept in the stone mortar.
2nd time:
Do the same as the 1st time with about 30 mL of distilled water.
3rd time:
Do the same as the 1st time with about 30 mL of distilled water.
After 3 times of extraction, you can use filter papers (takes about 1 to 1.5 hour)
or centrifuge (5000rs/m for 10 minutes) to remove the remained grounds out of
the extract solution.
After removing remained grounds, extracted protein solution will be poured into
volumetric flask of 100mL. Then, distilled water is added into the volumetric flask
to reach the marked level. This is your original protein solution or 100-diluted
solution.
Figure 1.1
Before centrifuging
Figure 1.2
Figure 1.3
Centrifuge
After centrifuging
0.5
1.0
1.5
2.0
2.5
10
9.5
9.0
8.5
8.0
7.5
50
100
150
200
250
After finishing making test tubes of protein solution followed the table above, you
continue to make test tubes of standard protein solutions followed the table below.
Step 3: 10 test tubes are numbered respectively from 1 to 10.
Tube number (mL)
Protein solution
0.4
0.4
0.4
0.4
0.4
0.4
0.4
0.4
10
0.4
0.4
Original solution
(100-diluted)
Sample solution
(10,000-diluted)
Solution C
Shake each tube well and also keep them for 10 minutes
Folin-Ciocalteu
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
Shake each tube well and also keep them for 10 minutes
Distilled water
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
Shake the tubes well, keep them for 5 minutes and measure the A750nm
Figure 1.4
Figure 1.5
50
10
OD
OD
Protein concentration
(g/mL)
4.2
Step 3: Click insert bar Black mark all table Choose Insert scatter
scatter
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4.3
EXPERIMENT 2
Exploring the enzymatic activity of bromelain
1. Principle
Bromelain is an enzyme classified in group of protease, and it is present much in
pineapple. To determine the enzymatic activity of bromelain, experimenter often
uses this enzyme to catalyze the degradation of substrate such as hemoglobin and
casein. This reaction produces peptides and amino acids, including tyrosine.
Tyrosine can be quantified colorimetrically after adding Folin-Ciocalteu reagent.
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Equipment
+ Test tubes
+ Pipettes (1mL, 2mL, and 5mL) and pump
+ Beakers (500mL, 250mL and 100mL)
+ Falcons (50mL)
+ Filter paper (11mm)
+ Spectrophotometer
+ Centrifuges
2.2
Chemicals
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3. Practical procedures
3.1 Preparation of sample
Step 1: Choose fresh, moderately ripe pineapple to discard, cut into the pieces
and then pulverize it by blender or stone mortar.
Step 2: Use filter papers (takes about 1 to 1.5 hour) or centrifuge (5000rs/m for
10 minutes) to remove the remained grounds out of the pineapple juice.
3.2
Figure 2.1
Figure 2.2
Pineapple
Step
2% Casein in the
phosphate buffer
Tube of tyrosine
Tube of sample
Tube of water
(mL)
(mL)
(mL)
2.5
2.5
2.5
0.2M HCl
0.5
0.5
0.5
Tyrosin solution
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Pineapple solution
containing bromelain
0.2
0.2
0.2
Step 2: Shake each tube well and keep them still for 10 minutes at 250C, then
filter the tubes.
Step 3: Transfer 2mL of filtered solution of each tube into new tubes
Step 4: Add 5mL of 0.5M NaOH solution and 1mL of Folin-Ciocalteu reagent
Step 5: Shake the tubes well, then put the tubes at the stand for 15 minutes and
measure the A578nm or A620nm.
Figure 2.3
White precipitate after adding tricloracetic 5%
Figure 2.4a
Figure 2.4b
Tube of water
Tube of sample
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Figure 2.4c
Tube of Tyrosine
450.ODM 1 x1
(UI / mL)
ODT
10 x2
Where:
x1: Tyrosine solution (mL)
x2: Enzyme solution (mL)
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EXPERIMENT 3
Soluble carbohydrate quantification applying Anthrone assay
1. Principle
In the anthrone assay, carbohydrate is dehydrated by using concentrated sulfuric
acid to form furfural, which in turn condenses with anthrone (10-keto-9,10
dihydroanthracene) to form bluish-green complex which can be measured
colorimetrically at wavelength of 620-630 nm by spectrophotometer. This a rapid
and convenient assay for determination of hexoses, aldopentose and hexuronic
acid, either in the free from or in form of polysaccharide. In this assay, the reagent
preparation does not require the addition of distilled water, and anthrone is directly
dissolved at a 2% concentration in concentrated sulfuric acid. This acid is powerful
dehydrating agent involved in dehydrating sugars leading to formation of furfural,
which condenses with anthrone to give the colored product.
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Equipment
Chemicals
+ Alcohol 90oC
+ Alcohol 80oC
+ Anthrone reagent
+ 0.01% Glucose solution
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3. Practical procedures
3.1 Preparation of sample
Step 1: Take 2g of pulverized raw material and put it into the beaker of 50mL.
Step 2: Add 10mL of alcohol 90o into the beaker.
Figure 3.1
Banana
Figure 3.2
Alcohol
Figure 3.3
Anthrone reagent
Step 3: Put the beaker in the water bath at 80oC. Using the stirring rod to stir the
solution well during the heating process.
Step 4: Get the solutes in alcohol by filtering the cloth.
Step 5: Add 10mL of alcohol 80o into the beaker of grain and do the same at step
3 and 4. Alcohol can be vaporized naturally or by providing heat slight to the
beaker. Do step 5 2 times.
Step 6: Dissolve the extracted soluble carbohydrate in 50mL of water to make
50-diluted solution by using the volumetric flask. If there is the sediment in
sample, let it settle down.
Step 7: Dilute the 50-diluted solution 100 times to get 5,000-diluted solution
because the amount of carbohydrate is unknown. If the carbohydrate content very
high, the solution need to be diluted more until the measured point is in range of
calibration curve.
Step 8: The sugar solution is taken to do color-forming reaction.
3.2
Color-forming reaction
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Tube number
Sample (mL)
The concentration in each
tube
10
11
Figure 3.4a
Figure 3.4b
Adding Anthrone reagent
Step 3: Stir the solution very slowly by a glass stick. Then boil all tubes in hot
water for 7.5 minutes. After that, put all tubes in cool water immediately.
Step 4: Finally, measure the A630nm.
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Figure 3.5a
Figure 3.5b
After Color-forming reaction (1)
Figure 3.5c
Figure 3.5d
After Color-forming reaction (2)
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EXPERIMENT 4
Quantitative determination of calcium in powdered milk
1. Principle
Oxalate ammonium will precipitate all calcium ion in any solution when the
experimenter set up all following conditions severely:
+ pH of solution environment is greater than 4
+ The hot, saturated (COONH4)2 solution is filled only one time into the sample
containing calcium ion.
+ Freeze the solution immediately after the solution has heated up for 1 minute
This method can be used for calcium quantification in milk, blood sample, urine,
food, etc. and the reactions are performed as following:
Ca2+ + (COONH4)2 2NH4+ + Ca(COO)2 (1)
Ca(COO)2 + H2SO4 CaSO4 + (COOH)2 (2)
5(COOH)2 + 2KMnO4 + 3H2SO4 K2SO4 + 2MnSO4 + 10CO2 + 8H2O (3)
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Equipment
+ Racemic crucibles
+ Muffle furnace
+ Desiccator
+ Burette
+ Erlenmeyer flasks (250mL)
+ Beakers (100mL)
+ pH meter
+ Filter papers (11mm)
2.2
Chemicals
+ Powdered milk
+ Saturated (COONH4)2
+ Concentrated HCl
+ Methyl red
+ 0.1M NH4OH
+ Acetic acid
+ Saturated Calcium chloride
+ 1N H2SO4
+ 0.02N KMnO4
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3. Practical procedures
3.1 Preparation of sample
Step 1: Pretreat milk sample of 0.5g by burning them with absolute ethanol or
dehydrate them with concentrated sulfuric acid.
Figure 4.1
Pretreating powdered milk with absolute ethanol
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Step 4: Adjust the pH of the solution to from 5 to 5.2 by acetic acid of weak
concentration. At that point, the solution color is orange-pink. Due to the
instrumental error, each pH meter will give a different value of pH. Therefore,
observe the color of solution is also very important. Do not over-rely on any type
of machine.
Figure 4.2a
Figure 4.2b
Sample solution after adjusting pH
Step 5: While heating theses beakers by water bath, stirring these solutions and
fill 2-3mL of saturated (COONH4)2 solution.
Figure 4.3a
Figure 4.3b
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Step 6: Continue to provide heat to these beakers and then mix these solution
well for 30 seconds.
Step 7: Put these beakers in cool basin of water immediately.
Step 8: Keep the beaker in the basin of water for about 30 minutes.
Figure 4.4
Keeping the beaker in the
basin of water
Figure 4.5a
Figure 4.5b
Filtering the precipitate
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Step 10: Use distilled water to wash the filter paper to know whether (COO)22ions are all eliminated or not. To check it, we use saturated calcium chloride
solution.
Figure 4.6
Using saturated calcium chloride solution to check the remained ion (COO)22
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Figure 4.7a
Figure 4.7b
The color of sample solution after checking in case of remaining ion (COO)22
Step 11: Collect precipitate retained by filter paper and put them into Erlenmeyer
flask
Step 12: Add 20mL of 1N sulfuric acid solution into each Erlenmeyer flask and
heat them in water bath with temperature of 70oC for 1 minute.
Step 13: Titrate the solution with 0.02 N potassium permanganate solution to
determine the concentration of (COO)22- ion in the solution.
Figure 4.8
Figure 4.9
After titrating
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Step 14: Use the rule of three to calculate the amount of calcium ion in the
solution and in the sample.
4. Results and data analysis
Step 1: Base on the reaction (3):
Mn7+ + 5e Mn2+
The relation between the concentration (M) and the concentration (N) is:
=
(mol)
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V: the volume (L) of 0.02 N KMnO4 that is used to determine the quantitative
measurement
m: the weight of the powdered milk (m=0.5g)
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EXPERIMENT 1
1. Why do we need to add Folin-Ciocalteu reagent into protein solution?
2. What is the principle that spectrophotometric method based on?
3. What is the role of Albumin in this experiment?
4. What are two reactions used for developing intensely blue colour?
5. Why do we need to make blank solution?
6. What is the disadvantage of Hartree-Lowry method?
7. Why the amount of protein of interest need to be firstly considered before choosing
quantification assay?
8. What will you do if you have to quantify protein A but you cannot buy the
commercialized standard protein A?
9. Student A says that he can insert any value of optical density in the equation of
standard curve to determine the corresponding value of protein content. Does the
student say it right or wrong? Explain why?
10. Student B says that in this practical, he can only determine relatively the total
protein content in extracted protein solution. Does the student say it right or wrong?
Explain why? Assuming that he is right, what will he do firstly before he want to
quantify mostly exactly his proteins of interest?
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EXPERIMENT 2
1. What fruit was bromelain extracted from? What is the main function of bromelain?
2. What are crucial factors which can have effects on enzymatic activity?
3. Are fixed times important for exploring/studying the enzymatic activity? Explain
why.
4. Why is trichloroacetic acid added into tube of Tyrosine and tube of water
immediately right after putting casein solution into the test tubes while with respect
to tube of sample, trichloroacetic acid is added at the end of the process?
5. Why shouldnt we add Tyrosine into tube of sample?
6. How can the activity of enzyme be determined in this experiment?
7. Student A says that when doing this practical, he can prepare the tubes of tyrosine
and tubes of water first and then measure the optical density in order to save time.
Does he says it right or wrong? Explain why.
8. Student B says that the more he add bromelain, the faster the catalytic reaction
occurs. Does he says it right or wrong? Explain why.
9. Student C says that if he can purify and recover the bromelain used in this
practical, he can continue to use it for the catalytic reaction next time in order to save
the enzyme. Does he says it right or wrong? Explain why.
10. Student D says that there is a paradox in enzymatic activity. He supposes that
protease can degrade itself because it is also a protein. Does he says it right or wrong?
Explain why.
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EXPERIMENT 3
1. What is the solvent of Anthrone reagent?
2. When the dried samples are used, why we need to use the fewer amounts
compared to the raw ones.
3. Why alcohols are used to extract carbohydrate?
4. How long does Anthrone reagent can be kept for using?
5. Why anthrone reagent needs to be kept cold before using?
6. Why test tubes must be put in ice-water beaker/container when adding Anthrone
solution?
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EXPERIMENT 4
1. What is the role of Oxalate ammonium?
2. Why do we need to use Methyl Red?
3. Why do we use acetic acid to adjust pH instead of strong acid such Chlorohydric
acid or Sulfuric acid?
4. Why dont prepare Potassium Permanganate solution before lab work so far?
5. Why do we need to eliminate all the (COO)22- ion out of the calcium?
6. Why does powdered milk must be pre-treated before heating in the muffle furnace?
7. Student A says that Ca2+ is an analyte and KMnO4 is a titrant. Does he says it right
or wrong? Explain why.
8. Why calcium chloride solution must be saturated?
9. Why dont we use non-heated powdered milk to quantify calcium content?
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36
EXPERIMENT 1
Folin-Ciocalteu reagent
Weigh 100g Sodium tungstate and 25g Sodium molybdate. Then, add 700mL distilled
water and 50mL concentrated orthophosphoric acid (83-85%). Stir well and add
100mL of concentrated chlohydric acid (37-27%). Stir well and reflux for 10 hours.
After refluxing, add 150g of lithium sulfate into the solution and wait until it
completely dissolved. Stir well and then add 10mL bromine solution of 3-5 drops of
pure bromine liquid. Finally, reflux for 30 minutes in the fume hood and cool the
solution at room temperature.
If the solution is not clear, filter them. The well-made solution has yellow-orange
color. If the solution turns into yellow-green, it cannot be used. Keep Folin-Ciocalteu
reagent at 4oC (in the fridge). This solution can be kept and used within 1 year.
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EXPERIMENT 2
Standard L-tyrosine solution
Get 45mg of L-tyrosine and dissolve in 100mL of solution of 0.2M HCl.
1M KH2PO4 buffer solution
Get 13.6g of KH2PO4 and dissolve it in 100mL of water.
2% Casein solution
Get 2g of casein, 36g urea and 8mL of 1M NaOH to dissolve it in 40mL of distilled
water. Keep the solution at 250C for 60 min, add 10mL of 1M KH2PO4 buffer solution.
Then, adjust the pH to 6 with 2M HCl. Finally, add distilled water to the point of
100mL and keep it in the fridge.
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EXPERIMENT 3
0.01% Glucose solution
Take 0.01g of glucose that has dried in the desiccator, then dissolve in 100ml of
water.
Anthrone reagent
Dissolve 2g of anthrone in 1 L of concentrated sulfuric acid. Then, keep anthrone
solution in the fridge. This reagent cannot be used after 48 hours.
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EXPERIMENT 4
Methyl red indicator
Dissolve 0.02 g in 60 mL of absolute ethanol and 40 mL distilled water.
40
Be careful with chemicals used in the lab due to their high toxicity, irritation,
corrosion or flammability.
The electrical equipment, including hot plates, stirring motors, and highvoltage power supplier present special hazards.
The accident may happen easily to anybody who do not follows the lab safety
regulations.
Be familiar with the location and use of standard safety features in your
laboratory.
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42
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References:
1) Arti Nigam and Archana Ayyagari - Lab Manual in Biochemistry:
Immunology and Biotechnology Page 33.
2) Gary Walsh - Proteins: Biotechnology and Biochemistry. Table 3.2
Page 93,94 - Chapter 3: Protein purification and characterization.
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