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Experiment No. 1
EXTRACTION AND CHARACTERIZATION OF PROTEINS

Jns Jacob Berzelius discovered proteins in 1838. They are one of the classes of
biological macromolecules, alongside polysaccharides and nucleic acids that make up
the primary constituents of living things.

Proteins are high molecular weight organic compounds with very complex
structures containing may different kinds of simple substances called amino acids joined
by peptide bonds. In addition, the side chains of various amino acids in the polypeptide
may interact, intrachain or interchain, resulting in many different physical and chemical
properties of proteins. Their functions range from the enzymes that carry out the
numerous metabolic processes of the cell to structural components that provides cells
their structures and organization.

Protein extraction involves a series of processes intended to isolate a single type


of protein from a complex mixture. Protein purification is vital for the characterization of
the function, structure and interactions of the protein of interest. The starting material is
usually a biological tissue or a microbial culture. The main goal in protein extraction is to
abstract the biomolecule efficiently, causing little degradation. Secondary but equally
important goals are to preserve the proteins as much as possible and to create an
accurate representation of all proteins present in the original tissue. A variety of protein
extraction methods have been developed. Common techniques used in these methods
include grinding, homogenizing, centrifugation, salting out, dialysis, and ultrafiltration.
The choice of technique/s depends on the type of tissues and cell materials, how the
sample has been treated, and the proteins that are to be extracted. Similarly, the reagent
to be used for protein extraction is also dependent on the aforementioned factors.

One of the most useful parameters used in the study of proteins in solution is the
concentration of protein as this is necessary in subsequent quantitative
determination
of the activity of the protein. The concentrations of protein in biological extracts can be
estimated using spectrophotometric methods. Bradford assay is a method based on the
binding of Coomassie brilliant blue dye to proteins under acidic conditions. The protein-
dye complex causes a shift in the dyes wavelength of maximum absorption from 465 nm
to 595 nm. By comparing the absorbance of a solution containing an unknown amount of
the protein dye complex to the absorbance values of some standard solutions having
known concentrations, the concentration of protein in the sample can be estimated. The
Bradford assay has been found useful in determining protein concentrations in the range
1-20 g/ml. The Warburg-Christian method, on the other hand, is more suitable for semi-
quantitative analysis of biological samples and is generally applied in detecting protein
concentrations in the range of 20-3000 g/ml. The method makes use of the maximum
absorbance of tyrosine and tryptophan residues at 280 nm for estimating total protein
content and the strong absorption of nucleic acids at 260 nm.
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Objectives

Upon completion of the experiment, the student should be able to:


1. isolate at least two (2) proteins from biological sources such as invertase from
yeast, casein from milk, albumin from egg and globulin from squash seeds.
2. describe the methods employed for protein extraction.
3. assess the activity of invertase extract using Benedicts test.
4. apply spectrophotometric methods in quantifying extracted proteins.

Methodology

Materials
95% ethanol 0.1 M HCl
0.3 M sucrose Benedicts reagent
2% glucose Bradford reagent
2% fructose mortar & pestle
2.0 M HCl evaporating dish
2.0 M M NaOH filter paper
10% CH3COOH pH paper
0.01 M NaOH ice
2.0 M CH3COOH cheesecloth
saturated (NH4)2SO4 solution Bakers yeast
hexane Fine sand
petroleum ether eggs
0.1 M acetate buffer pH 5 milk
5.0% NaCl squash/cucumber seeds
0.9% NaCl cuvettes
1.0 % standard casein solution watch glass
1.0 % standard globulin solution Eppendorf/centrifuge tubes
1.0 % standard BSA solution

Equipment
top loading balance food processor/blender
centrifuge UV-Vis spectrophotometer
hot plate with magnetic stirrer

Procedure
I. Extraction of Proteins
A. Invertase from Yeast
1. Immerse a clean 50-ml beaker and another beaker with 150 ml 95% ethanol in
an ice bath.
2. Grind 20 g bakers yeast with 5 g of sand in a clean mortar and pestle until a
fine powder is obtained. Add 20 ml hexane to the fine powder.
3. Add 60 ml water in 5 ml portions and continue grinding for another 10-15 mins.
4. Filter the ground yeast through cheesecloth to obtain a cell-free extract.
5. Centrifuge the filtrate at 6000 rpm for 5 minutes. Discard the sediment. Repeat
centrifugation step until supernatant is relatively clear. (Use longer time if
necessary.)
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6. Pour the supernatant into the pre-cooled beaker. Slowly add the cold 95%
ethanol with a volume equivalent to 4 times that of the extract. Do not stir. Set
aside in an ice bath until precipitation occurs.
7. Centrifuge the resulting suspension at 6000 rpm for 5 mins.
8. Weigh a clean and dry (small) watch glass up to the hundredth of a gram.
9. Discard the supernatant after centrifugation and transfer the crude extract to
the pre-weighed watch glass. Air dry under the hood.
10. Determine the weight of the extract using weighing by difference. Have the
crude extract assessed by the instructor before proceeding to the next step.
11. Dissolve the precipitate in enough 0.1 M acetate buffer pH 5 to make 10%
(w/v) solution.
12. Place the solution in a vial with a tight lid, label and keep in the refrigerator if it
cannot be used for part II in the same laboratory period.

B. Albumin from Egg


1. Separate the egg white from the yolk of one medium sized egg and obtain 30
mL in a 50-mL graduated cylinder.
2. Stir the egg white with a stirring rod and add 3 mL of 1.0 M HOAc dropwise.
3. Filter the mixture through cheesecloth by stirring with a glass rod to speed up
the passage of the filtrate and squeeze the cheesecloth to break the
membranes. Collect the filtrate in a 250-mL beaker.
4. Add an equal volume of saturated (NH4)2SO4 solution to the filtrate.
5. Allow the mixture to stand for 30 minutes.
6. Centrifuge the mixture at 3000 rpm for 5 minutes and discard the precipitate.
7. Transfer the supernatant into a 250-mL Erlenmeyer flask.
8. Add (NH4)2SO4 buffer to the clear yellow supernatant while continuously stirring
until turbidity persists.
9. Store the mixture in the refrigerator until the next laboratory period.
10. Centrifuge the mixture and discard the supernatant.
11. Air dry the albumin extract under the hood.
12. Determine the weight of the dry precipitate using weighing by difference. Have
the crude extract checked by the instructor.
13. Place the precipitate in a vial with a tight lid, label and store in the refrigerator if
it cannot be used for part II in the same laboratory period.

C. Casein from Milk


1. Weigh 20 g non-fat powdered milk in a weighing paper.
2. Dissolve the milk in 200 ml warm water in a 250-ml beaker. (If using fresh
liquid milk, measure 200 mL directly.)
3. Heat the milk solution to 40 C.
4. Add 10 ml 10% acetic acid solution while stirring the mixture slowly and briefly.
Avoid adding excess acid.
5. Centrifuge the mixture at 3000 rpm for 10 minutes and discard the
supernatant.
6. Wash the curd by resuspending it in enough cold 95% ethanol. Mix thoroughly
to wash the residue. Decant the ethanol washings. Wash the residue with
ethanol repeatedly until the ethanol washing is clear.
7. Weigh a piece of filter paper up to the hundredth of a gram.
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8. Transfer the moist residue into the pre-weighed filter paper, wash with acetone
into a funnel and air dry under the hood. Determine the mass of the crude
extract by weighing by difference and have it assessed by the instructor.
9. Place the precipitate in a vial with a tight lid, label and store in the refrigerator if
it cannot be used for part II on the same laboratory period.

D. Globulin from Squash/Cucumber Seeds


1. Immerse two clean 250-ml beakers and 50-ml each of 2M NaOH and 2M HCl
in an ice bath.
2. Wash 25 g of seeds thoroughly with distilled water. Pat dry and press between
filter paper to remove most of the water.
3. Grind the seeds into a powder with intermittent pulses (turning the equipment
on and off at 30-sec interval) in a food processor or blender.
4. Transfer the powder into a beaker and add 50 ml hexane or petroleum ether.
Mix vigorously using a stirring rod.
5. Filter the mixture through a cheesecloth. Collect the residue in an evaporating
dish and air dry the material under the hood for 30 mins.
6. Place the air-dried residue into the pre-cooled 250-ml beaker and add 50 ml
water. Place the beaker on a magnetic stirrer and stir the mixture for an hour at
RT.
7. Adjust the pH of the mixture to 9 by adding cold 2 M NaOH drop by drop. Stir
for another hour at RT with occasional pH checks and adjust the pH to 9, if
necessary.
8. Filter the material through cheese cloth. Collect the filtrate into the 2 nd pre-
cooled 250-ml beaker.
9. Adjust the pH of the filtrate to 4.5 by adding cold 2 M HCl. Let the mixture
stand in an ice bath for an hour or until precipitation is complete.
(Alternatively, the air-dried residue from step 4 can be processed by salt
extraction with 100 mL 5% NaCl but this will require stirring the mixture for 4 h
at RT, instead of using steps 6,7,8)
10. Centrifuge the suspension at 3,000 rpm for 5 mins.
11. Weigh a filter paper up to the hundredth of a gram.
12. Collect the residue in the pre-weighed filter paper and wash with a minimal
amount of acetone into a funnel. Air dry under the hood. Determine the mass
of the crude extract by weighing by difference and have it checked by the
instructor.
13. Place the precipitate in a vial with a tight lid, label and store in the refrigerator if
it cannot be used for part II on the same laboratory period.

II. Characterization
A. Activity Assay for Invertase
1. Take out the stock 10% (w/v) invertase solution from the refrigerator and immerse
it in an ice bath.
2. Prepare a set of four test tubes according to the table below:
Volume, ml
Test tube 0.3 M sucrose H2O Acetate buffer 2% glucose 2% fructose
1 3.0 2.0 3.0 --- ---
2 3.0 4.0 3.0 --- ---
3 --- 4.0 3.0 3.0 -
4 --- 4.0 3.0 --- 3.0
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3. Place tubes 1 and 2 in a water bath maintained at 37C. Allow to equilibrate for
5-10 mins.
4. Add 2 ml of invertase extract to tube 1. Keep tubes 1 and 2 in the water bath for an
additional 6 mins.
5. Stop the reaction by adding 2 ml 10% NaOH each to tubes 1 and 2.
6. Add 1 ml Benedicts reagent to tubes 1 - 4 and heat in a boiling water bath for
5 10 mins. Record the amount of time that elapsed until a brick-red precipitate
is obtained.

B. Quantification of Protein Concentration by Spectrophotometry


1. Warburg- Christian Method
Sample preparation (for Expt. 1, Parts II-B1 and B2 and for Expt. 2
i. Prepare 20.0 mL of 1% (w/v) solution of albumin in 0.9% NaCl.
ii. Prepare 20.0 mL of 1% (w/v) casein solution using 0.01 M NaOH.
iii. Prepare 20.0 ml of 1% (w/v) globulin solution in distilled water.
Reduce volumes to be prepared based on available mass of protein extract
but always consult your instructor first.
Always store unused protein solutions in properly labeled, covered containers
in the refrigerator until Expt 2 is completed..
Spectrophotometric Analysis
i. Measure the absorbance of the protein extracts at 280 and 260 nm against
the following blanks: 0.9% NaCl for albumin, 0.01 M NaOH for casein and
distilled water for globulin.
ii. Compute the A280/A260 ratio and refer to the table below to estimate the %
nucleic acid in the protein extract. Estimate the purity of the protein isolate by
subtracting the corresponding % nucleic acid from 100%.
A280/A260 % Nucleic Acid A280/A260 % Nucleic Acid
1.75 0.00 0.87 5.00
1.63 0.25 0.85 5.50
1.52 0.50 0.82 6.00
1.40 0.75 0.80 6.50
1.36 1.00 0.78 7.00
1.30 1.25 0.77 7.50
1.25 1.50 0.75 8.00
1.16 2.00 0.73 9.00
1.09 2.50 0.71 10.00
1.03 3.00 0.67 12.00
0.98 3.50 0.64 14.00
0.94 4.00 0.62 17.00
0.90 4.50 0.60 20.00
iii. Calculate the protein concentration in the crude extracts using the following
equation:
Protein concentration (mg/ml) = {1.55 A280 - 0.76 A260} x dilution factor
2. Bradford Assay
i. Set up 12 test tubes and add protein standards and protein extracts according
to the table on the next page. (Tube 1 is used as blank and tubes 2 -6 for the
construction of a standard curve for protein quantitation. Tubes 7 -12 are
duplicates of the three different concentrations of the isolates.)
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Volume, ml
Test tube 1 2 3 4 5 6 7 8 9 10 11 12
Standard
0.0 0.2 0.4 0.6 0.8 1.0 --- --- --- --- --- ---
protein*
Protein
--- --- --- --- --- --- 0.3 0.3 0.5 0.5 0.7 0.7
extract
Distilled
4.8 4.6 4.4 4.2 4.0 3.8 4.5 4.5 4.3 4.3 4.1 4.1
H2O
Bradford
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
reagent
*standard solution to be used should be of the same protein as that of the extract
ii. Add enough distilled water to each tube according to the table.
iii. Add 0.2 ml Bradford reagent to each test tube and mix thoroughly.
iv. After 5 mins, read the absorbance of each tube at 595 nm. The absorbance
readings should be taken within 1 h after the addition of the dye.
v. Construct a calibration curve and determine the protein concentration of the crude
extract.
vi. If the crude extracts have absorbance readings outside the range established by
the standard curve, dilute the extracts with water and redo the absorbance
measurements at 595 nm. Consider the dilution factors when calculating for the
actual protein concentration of the extracts.

Waste Disposal
1. Collect all solid wastes in a garbage bag and dispose in the trash bin.
2. Discard mixtures from the two assays in the appropriate waste containers under the
hood.
3. Place all acid and alkaline wastes into their respective disposal jars.
4. Discard hexane- or petroleum ether-containing wastes into the organic wastes jar.
5. Dispose all other waste solutions into the sink. Flush with ample amounts of running
water.
6. Cheesecloths may be washed with detergent. Reuse if necessary.

References

Biochemistry Laboratory Manual, Quezon City: UP Diliman, 2002.


Boyer, R.F. Modern Experimental Biochemistry. Benjamin Cummings, 1993.
Curr Protoc Mol Biol. Nov. 2006.
http://www.biochem.ucl.ac.uk/chen/protocols
http://www.ehow.com/facts_7362247_protocol-protein-extraction.html
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NAME
SECTION DATE
INSTRUCTOR

PRE-LAB SHEET FOR EXPERIMENT 1


EXTRACTION AND CHARACTERIZATION OF PROTEINS

1. Draw schematic diagrams for the extraction of the protein(s) assigned to your
group.
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2. List down all the classes of proteins based on function/s.

3. What is the composition of the Bradford reagent? How is a color change obtained
when it binds to proteins?

4. What is a reducing sugar? Explain in terms of mutarotation of the hemiacetal.

5. Provide safety information from the MSDS of three hazardous reagents to be used in
the experiment. Use the format provided in the syllabus.
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NAME
SECTION DATE
INSTRUCTOR

POST-LAB SHEET FOR EXPERIMENT 1


EXTRACTION AND CHARACTERIZATION OF PROTEINS

1. What principle is involved in the extraction of the following?


a. invertase

b. albumin

c. casein

d. globulin

2. State the purpose of each step in the extraction procedure. (For separation procedures
such as filtration, decantation and centrifugation, indicate what is being removed.)
a. Invertase from yeast
Grinding with sand
Addition of hexane
Gradual addition of water
Filtration using cheesecloth
Addition of cold 95% EtOH
Centrifugation

b. Albumin from egg


Separation of egg white from egg yolk
Dropwise addition of 1M HOAc
Filtration using cheesecloth
Addition of saturated (NH4)2SO4 & centrifugation
Second addition of (NH4)2SO4 and cooling
Centrifugation

c. Casein from milk


Warming
Gradual addition of 10% HOAc
Centrifugation
Washing with cold 95% EtOH
Washing with acetone
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d. Globulin from Squash Seeds


Grinding in a food processor
Addition of hexane or petroleum ether
Filtration through cheesecloth
Addition of water and stirring
Addition of cold 2M NaOH to pH 9, stirring & filtration
Addition of cold 2M HCl to pH 4.5, cooling
Centrifugation
Washing with acetone

3. Classify the proteins studied in this experiment based on function/s.


invertase casein
albumin globulin

4. Show sample calculations for the concentration and % purity of the protein(s) extracted
by your group. (Except if your group was assigned invertase, obtain data from another
group.)

5. Compare & contrast the Warburg-Christian method from the Bradford assay as protein
concentration assay techniques. Use your results to explain which technique is more
efficient.

6. Interpret the results of the invertase activity assay.