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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.
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
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.
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.
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.
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
NAME
SECTION DATE
INSTRUCTOR
1. Draw schematic diagrams for the extraction of the protein(s) assigned to your
group.
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3. What is the composition of the Bradford reagent? How is a color change obtained
when it binds to proteins?
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
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
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.