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 Sensitivity Mechanism Advantage Disadvantage

Bradford 150-750 µg/mL Based upon reversible, pH Moderately Alkaline
dependent binding of sensitive, pH/buffer will
coomassie brilliant blue G- easy and fast interfere
250 dye to protein. to run
Absorbance at 595 nm
Hartree- 30-150 µg/mL Combination of copper and Good Laborious.
Lowry phosphomolybdic/phospho sensitivity Detergent and
tungtic acid reacts chelating agent
quantitatively with protein. can interfere.
Absorbance at 750 nm
Biuret

Fluorescence 5-50 µg/mL Based on fluorescence Very Same

emission properties of aromatic amino sensitive, fast concentration
acid residue in the protein. and non- of different
destructive to proteins can
sample lead to
variation in
reading result
due to various
content of
aromatic
residue.
The purified source of the protein to be quantified should be chosen as the
standard one to reduce result’s errors. In case of the proteins of interest whose
sources have not been isolated, purified or commercially sold, the other proteins
having similar structures or coming from the same family protein should be
chosen as alternatives, instead to obtain a high similar color yield.

In this practical, Hartree-Lowry assay is used for protein quantification. Standard

protein of known concentration is used to construct calibration curve. Folin
– Ciocalteu reagent is added to the protein solutions to develop a color whose
intensity is measured colorimetrically. Albumin solution is selected as an
appropriate standard. Various known concentration of albumin solution are
mixed well with reagent to enhance color development. Two below reactions
account for intensely blue color development:

+ The coordination of peptide bonds with alkaline copper (biuret assay)

+ The reduction of the Folin – Ciocalteu reagent by tyrosine and tryptophan

residues in protein.

The advantage of Hartree-Lowry assay is its sensitivity, which is up to 100 times

greater than of the Biuret assay; however, more time is required when applying
Hartree-Lowry assay. Since extracted protein solution is a mixture of different
proteins whose tyrosine and tryptophan contents are variable, the color
development may changes even though their concentration are the same.
2. Equipment and chemicals
2.1 Equipment
+ Test tubes
+ Pipettes (1mL, 2mL, and 5mL) and pumps
+ Volumetric flasks (50mL, 100mL)
+ Beakers (50mL, 100mL)
+ Graduated cylinder (50mL)
+ Falcons (50mL)
+ Filter paper (11mm)
+ Spectrophotometer
+ Centrifuges
2.2 Chemicals
+ 0.1% Albumin solution
+ Solution A
+ Solution B
+ Solution C
+ Folin-Ciocalteu reagent
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
(about 20mL) 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 filtered soybean 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, 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.
You need to make 10,000-diluted solution by dissolving 1mL of 100-diluted
solution in 99mL of distilled water.

Figure 1.1 Figure 1.2 Figure 1.3

Before centrifuging Centrifuge After centrifuging

3.3 Making standard curve and quantifying protein content of sample

Step 1: 6 test tubes are numbered respectively from 1 to 6.

Step 2: The 0.1% albumin solution is diluted with the different amount of
water to make the protein solutions with different concentrations (0, 50, 100,
150,
200, and 250μg/mL).

To get the expected result, we must follow the procedure in the table below.

Tube Number 1 2 3 4 5 6
0.1% Albumin solution (mL) 0 0.5 1.0 1.5 2.0 2.5
Distilled water (mL) 10 9.5 9.0 8.5 8.0 7.5
Shake the tube well
The concentration of each tube (μg/mL) 0 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) 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’

Protein solution 0.4 0.4 0.4 0.4 0.4 0.4
Original solution
0.4 0.4
(100-diluted)
Sample solution
0.4 0.4
(10,000-diluted)
Solution C 2 2 2 2 2 2 2 2 2 2
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

Before adding reagent After adding reagent
4. Results and data analysis

4.1 Result table

Tube number 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’

OD
ΔOD
Protein concentration
0 50 100 150 200 250 x Y
(μg/mL)

4.2 Using Microsoft excel to draw the standard curve

Step 1: Open excel

Step 2: Insert data

Step 3: Click insert bar  Black mark all table  Choose Insert scatter 
scatter

Step 4: Obtain the scatter graph without standard curve

Step 5: Click chart element  choose trend line  more option

 Step 6: Choose “display equation” on chart and “display R-squared value” on
chart

Step 7: Name the graph and put the unit

4.3 How to calculation the gram of protein that contains in 100 gram of
soybean

Step 1: Use the obtained equation to calculate the protein concentration of

sample corresponding to its measured optical density (OD).

Step 2: Reject the point that is out of range of the graph.

Step 3: Convert the unit from microgram into gram

Step 4: Use the rule of three to determine the protein concentration

containing in 10,000-diluted sample solution and 100-diluted sample solution.

Step 5: Determine the gram of soybean protein in 100 mL of 100-dilulted

sample solution (mProtein = 100×con100-diluted) then deduce the ratio of protein
content in 5 gram of soybean (mProtein /5)
 EXPERIMENT 2
Exploring the enzymatic activity of bromelain
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 colorimertrically after adding Folin – Ciocalteu

reagent.
 1. Equipment and chemicals
1.1 Equipment
+ Test tubes
+ Pipettes (1mL, 2mL, and 5mL) and pump
+ Beakers (500mL, 250mL and 100mL)
+ Falcons (50mL)
+ Filter paper (11mm)
+ Spectrophotometer
+ Centrifuges
1.2 Chemicals
+ 0.5M NaOH solution
+ 0.2M HCl solution
+ 5% Trichloroacetic acid solution
+ 2% Casein solution
+ Tyrosine solution
+ Folin-Ciocalteu reagent
 2. Practical procedures
2.1 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.

Figure 2.1 Figure 2.2

Pineapple Pineapple extract after centrifuging

2.2 Survey the enzymatic activity

Step 1: You perform the experiment following the table below.

Tube of tyrosine Tube of sample Tube of water

Step (mL) (mL) (mL)
2% Casein in the
2.5 2.5 2.5
phosphate buffer
Keep the tubes at 350 C water bath for 5
minutes
5% Trichloroacetic acid 5 0 5
0.2M HCl 0 0.5 0.5
Tyrosin solution 0.5 0 0
 Pineapple solution
0 0.2 0
containing bromelain
Shake the tubes well at 350C water bath for
10 minutes
5% Trichloroacetic acid 0 5 0
Pineapple solution
0.2 0 0.2
containing bromelain

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 Figure 2.4c

Tube of water Tube of sample Tube of Tyrosine

 3. Results and data analysis

The activity of bromelain enzyme is determined depending on quantity (μg) of

tyrosine produced from casein degradation under catalysis of enzyme in 1mL of
solution or 1mg of bromelain mixture for 1 minute.

The enzymatic activity is calculated by the formula below:

450. ΔOD M 1 X 1
10 X 2 (UI/mL)
. .
Δ ODT
Where:

x1: Tyrosine solution (mL)

x2: Enzyme solution (mL)

 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 measure
colorimetrically at wavelength of 620-630 nm by spectrophotometer. This is 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.

The accuracy of the reaction is based on the cleanness of equipment, the purification
of the reagent, especially for sulfuric acid and the constant temperature during the
boiling time.
2. Equipment and chemicals
2.1 Equipment

+ Volumetric flasks (50mL and 100mL)

+ Beakers (50mL and 100mL)
+ Stone mortar
+ Filter paper (11mm)
+ Test tubes
+ Pipettes (1mL and 10mL)
+ Spectrophotometer

2.2 Chemicals

+ Alcohol 90oC
+ Alcohol 80oC
+ Anthrone reagent
+ 0.01% Glucose solution
 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 Figure 3.2 Figure 3.3

Banana Alcohol 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 100mL of water to make

100-diluted solution by using the volumetric flask. If there is the sediment in
sample, let it settle down.

Step 7: Dilute the 100-diluted solution 100 times to get 10,000-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

 Tube number 1 2 3 4 5 6 7 8 9 10
0.01% Glucose (mL) 0 1 2 3 4 5
Distilled water (mL) 5 4 3 2 1 0
Sample (mL) 5 5
The concentration in each
0 0.1 0.2 0.3 0.4 0.5 x Y
tube

Step 1: Put all tubes in the ice-water.

Step 2: Put slowly 10 mL of Anthrone reagent into each tube. Let the reagent flow
along the inside-surface of the tube.

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.

 Figure 3.5a Figure 3.5b

After Color-forming reaction (1)

Figure 3.5c Figure 3.5d

After Color-forming reaction (2)

4. Results and data analysis

Look at the guideline in experiment 1 to calculate the result

 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 the 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)

 2. Equipment and chemicals
2.1 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
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

Step 2: After the milk sample is almost

completely burned, it turns into black color.

Step 3: Put the pretreated milk samples

contained in racemic crucibles into the muffle
furnace to heat with temperature of 500oC for
15 minutes, counted from the time that
temperature of the muffle furnace reach
500oC.

Step 4: Let the milk ash sample cool down and put them into desiccator over
night before performing experiment.

3.2 Calcium quantification

Step 1: Take three milk ash samples contained in three separated racemic
crucibles out of the desiccator and add 5mL of distilled water, and then 5 drops
of concentrated chlohydric acid (HCl)

Step 2: Mix well and transfer these solutions separately into 3 different beakers
of 250mL to adjust pH.

Step 3: Add 10-15 drops of methyl red and carry out the neutralization by
ammonium solution.
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.

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 3mL of saturated (COONH4)2 solution.

Figure 4.3a Figure 4.3b

Sample solution after filling 2-3mL of saturated (COONH4)2

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

Step 9: Use filter paper to collect all the precipitate until there is no precipitate in
the solution. Repeat it until we collect it all (about 4 – 5 times)

Figure 4.5a Figure 4.5b

Filtering the precipitate

Step 10: Use distilled water to wash the filter paper to know whether (COO) 22-
ions are all eliminated or not. To check it, we use saturated calcium chloride
solution ( add into the solution in the flask)

Figure 4.6

Using saturated calcium chloride solution to check the remained ion (COO)22
 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

Heating in water bath After titrating

Step 14: 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:

𝑁
𝑀=
𝑛𝑒

Where ne is quantity of electron(s) that is/are used to transfer from reductive

substance to oxidative substance in the reaction.

Step 2:

+ The moles of 0.02 N KMnO4 that contains in 1 liter of solution is:

0.02
(mol)
5

+ The moles of 0.02 N KMnO4 that contains in V liter of solution is:

V×4×10-3 (mol)

+ Base on the reactions (1), (2), (3), we have:

nCa2+ = 5/2×nKMnO4 = 5/2×V×4×10-3 (mol) = 0.01V (mol)

+ The gram of Calcium that contains in (m) g of milk powder:

mCa2+ = 0.01V×40 (g) = 0.4V (g)

+ The gram of Calcium that contains in 100g of milk powder:

mCa2+= 0.4V×100/0.5 = 80V (g)

Where
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)

PART II: REVIEW QUESTIONS
 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?
 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 shouldn’t 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.
 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?
 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 don’t 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 don’t we use non-heated powdered milk to quantify calcium content?
 PART III: MAKING SOLUTION IN
BIOCHEMISTRY LAB
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.

0.1% Albumin solution

Get exact amount of 0.1g of albumin, then dissolve it in water to make 100mL of
solution.

Solution A
Get 2g of Na2CO3 and dissolve it in 0.1 M NaOH to make 100mL.

Solution B
Get 0.5g of CuSO4.5H2O and dissolve it in 1% Sodium Citrate to make 100mL

Solution C
This solution can only be used within a hour, and it is the mixture of solution A and
solution B at a rate 49:1. When the solution C loses its pale-blue color, it cannot be
used any more.
 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.

EXPERIMENT 3
1.1 % 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.
 EXPERIMENT 4

Methyl red indicator

Dissolve 0.02 g in 60 mL of absolute ethanol and 40 mL distilled water.
 PART IV: SAFETY REGULATIONS IN
LABORATORY
1. Safety in the laboratory
You have to comply with safety regulations in laboratory

 Be careful with chemicals used in the lab due to their high toxicity,
irritation, corrosion or flammability.
 Be careful with fragile, glass, sharp and potentially infectious equipment,
material or sample such as disposable pipets, needles, blood, bacteria…
 Waste chemicals, disposable equipment, and infectious agents must be
separated and discarded in suitable place in order to protect the working
condition in lab, public place and environment.
 The electrical equipment, including hot plates, stirring motors, and high-
voltage power supplier present special hazards.
 The accident may happen easily to anybody who do not follows the lab
safety regulations.
 Using protective eyewear and gloves is highly recommended when working
with hazardous chemicals or infectious agents
 Do not work alone in the laboratory.
 Understand properties of all chemicals to be used such as their
flammability, reactivity, toxicity, and proper disposal.
 Eating, drinking, and smoking are not allowed in laboratory.
 Unauthorized experiments are not allowed.
 Mouth suction should not be used to fill pipets or to start a siphons.
 Be familiar with the location and use of standard safety features in your
laboratory.
 2. The laboratory notebook and experiment reports

Lab-notebook for recording the procedural details, observation, and result

should be written clearly, concisely, orderly, and accurately.

The most readable notebook is one in which only the right hand pages are
used for record keeping. The left hand pages may be used for your own
notes, reminders, and calculation.

In the biochemistry lab, you must read the material very well at home
and write down the procedure in your notebook. This will be checked by
the lab instructor. If not, you will be asked to stop working and go out.

3. Cleaning laboratory glassware

You must clean the lab-glassware carefully and return them to their previous
place because:

 Many of the chemicals will be used in milligram or microgram amounts.

Any contamination, whether on the inner wall of a beaker, in a pipette,
or in a glass cuvette, could be a significant percentage of the total
experimental sample.

 Many chemicals and biochemical processes are sensitive to one or more

of the following common contaminants: metal ion, detergents, and
organic residues.

Glassware: many contaminants, including organics and metal ions adhering

to the inner wall of glass containers. Washing the glassware, including pipets,
with dilute detergent (0.5% in water) followed by five to ten water rinses is
probably sufficient for most purposes. The final rinse with the distilled or
deionized water. Metal ion contamination can be greatly reduced from
glassware by rinsing with the concentrated nitric acid followed by extensive
rinsing with purified water. Then, dry equipment is required for most
processes carried out in the biochemistry laboratory. The equipment is dried
naturally or dried by drying cabinet.
Glass cuvettes: never clean cuvettes or optically polished glassware with
ethanol, KOH, or other strong base, as this will cause etching. All cuvettes
should be cleaned carefully with 0.5% detergent solution, in a sonicator bath,
or in a cuvette washer.
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.
BIOCHEMISTRY
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

PART II: REVIEW QUESTIONS

PART III: MAKING SOLUTION IN BIOCHEMISTRY LAB

PART IV: SAFETY REGULATIONS IN LABORATORY

1
PART I. PRACTICAL EXPERIMENTS