Protocols

IDENTIFICATION OF PROTEIN (CASEIN)
Milk contains three different forms of globular proteins: caseins, lactalbumins and lactoglobulins.
Globular proteins fail to interact with themselves and form colloid suspensions more easily than
fibrous proteins Therefore globular proteins can be more easily purified Casein is a mixture of
three similar proteins, called the alpha, beta, and kappa caseins
The Biuret Test

This is one of the most general tests for proteins. When a protein reacts with copper (II) sulfate,
a Positive test is the formation of a copper complex which has a violet color.
The Ninhydrin Test
Amino acids with a free –NH2 group and/or proteins with amino group side chains react with
ninhydrin to give a purble-blue complex.
Isolation of Casein
1. To a 250-mL Erlenmeyer flask, add approximately 50 g of 2% milk. Record the mass to the nearest 1/100 th of a
gram. Heat the flask in a water bath while stirring the milk. When the milk temperature has reached 40 oC,
remove the flask from the water bath, and add 10 drops of glacial acetic acid while stirring.
2. Filter mixture by pouring it through 4 layers of cheesecloth held in a 100-mL beaker. Rinse the Erlenmeyer
flask with 0.1 M acetic acid if necessary. Remove most of the liquid from the solid (casein and fat) by
squeezing the cloth gently. Discard the filtrate.
3. Place the solid into a 100-mL beaker and add 40-mL of 95% ethanol. After stirring the mixture for 5 min.,
allow the solid to settle. Carefully decant the liquid containing the fat into a beaker. Discard the liquid.
4. To the residue, add 15-mL of 95% ethanol and10-mL diethylether. After stirring the mixture 5 min., collect
the solid by vacuum filtration using pre-weighed filter paper.
5. Allow the casein to dry, weigh it, and calculate the percentage of casein in the milk.
Absorbance Assay (280 nm)

Absorbance assays are fast and convenient, since no additional reagents or incubations are required. No protein
standards need be prepared. The assay does not consume the protein. The relationship of absorbance to protein
concentration is linear. Any non-protein component of the solution that absorbs ultraviolet light will interfere with
the assay. Cell and tissue fractionation samples often contain insoluble or colored components that interfere.
Principle
Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with
aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily
responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance therefore
factors such as pH, ionic strength, etc. can alter the absorbance spectrum.
Equipment
In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are
required.
Procedure
Conduct steps 1-4 for a very rough estimate. Conduct all steps if nucleic acid contamination is likely.
1 Warm up the UV lamp (about 15 min.)
2 Adjust wavelength to 280 nm
3 Calibrate to zero absorbance with buffer solution only
4 Measure absorbance of the protein solution
5 Adjust wavelength to 260 nm Biochemistry Laboratory Manual Chemistry
6 Calibrate to zero absorbance with buffer solution only Department Peru State College Peru
7 Measure absorbance of the protein solution. By Dr. Dennis Welsh
Colorimetric Assays
Bradford protein assay
This is the assay of choice in most cases due to its simplicity, scalability and sensitivity. The absorbance maximum
for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465nm to 595nm upon protein binding. Both
hydrophobic and ionic interactions stabilise the anionic form of the dye, causing a visible color change. Range: 1 to
20 micrograms (micro assay); 20 to 200 micrograms.
Requirements
(1) Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol and add
100 ml of 85% (w/v) phosphoric acid. Dilute to 1 litre when the dye has completely dissolved, and filter
through Whatman #1 paper just before use
(2) 1M NaOH
(3) Colorimeter
(4) Glass or polystyrene cuvettes.
A manual for biochemistry protocol
Jan-thorsten schantz series
http://brilliantbiologystudent.weebly.com/iodine-test-for-starch.html
Iodine Test for Starch

The Iodine Test for Starch is used to determine the presence of starch in
biological materials, Starch is a polysaccharide consisting of glucose units joined together
by glycosidicbonds The chains formed during the condensation reaction are either linear or highly
branched molecules.
The sole reagent required for the test is bench iodine solution (0.1 M
potassium triiodide solution)
Procedure
1. Add 10 cm3 of the liquid food sample to a clean, dry test tube.
2. Add about 5 drops of iodine solution to the test tube.
3. Note any colour changes
Observation Interpretation
No change (Iodine remains Starch is not present
brown)
Starch is present
A blue-black colour
develops
Emulsion (ethanol) test for Fats

This test is done to show the presence of lipids in a substance. The substance is first dissolved
in ethanol. This solution is then dissolved in water. If lipids are present in the mixture, it will
precipitates and forms an emulsion.
Explanation
 Lipids are insoluble in water and soluble in ethanol (an alcohol).
 After lipids have been dissolved in ethanol and then added to H2O, they will form tiny
dispersed droplets in the water. This is called an emulsion.
 These droplets scatter light as it passes through the water so it appears white and cloudy.
Process
 Add the food sample to 2 cm3 of ethanol, shake well.
 Allow to settle in a test tube rack for 2 minutes for food to dissolve in ethanol.
 Empty any clear liquid into a test tube containing 2 cm3 of distilled H2O.
 A MILKY-WHITE EMULSION is a positive result: lipid is present.
 If the mixture remains clear, there are no fats present in the sample.
http://igbiologyy.blogspot.com/2012/12/32-emulsion-ethanol-test-for-fats.html
Testing for Nucleic Acids

DNA and RNA are nucleic acids made of nucleotide subunits. One major difference between DNA and
RNA is their sugar: DNA contains deoxyribose, whereas RNA contains ribose. DNA can be identified
chemically with the Dische diphenylamine test. In this test, acidic conditions convert deoxyribose
to a molecule that binds with diphenylamine to form a blue complex. The intensity of the blue color is
proportional to the concentration of DNA.
Procedure 5: The Dische diphenylamine test for DNA

1. Obtain four test tubes and number them 1-4.
2. Add 2 ml of the Dische diphenylamine reagent to each tube and mix thoroughly.
3. Place the tubes in a boiling water-bath to speed the reaction.
4. After 10 min, transfer the tubes to an ice bath. Gently mix and observe the color of their contents as the
tubes cool.
Result : if the color turn blue DNA is present in a sample
Molar Solution:
Number of moles of solute per 1000 ml/1dm3 of solution.
To prepare a solution of H2SO4 in 70 ml water, the normality of which is 0.25
N.
Calculation:
To prepare a solution of 0.25 N H2SO4 in 70 ml of water.
Gram equivalent of acid = Molar mass ÷ Acidity
Calculations:
Equivalent weight of H2SO4 = 98/2 = 49
To calculate normality of stock solution = %age purity × 10 × density ÷ eq. weight
1.84 × 98 × 10 ÷ 49 = 36.8
To calculate required volume:
Applying formula N1V1 = N2V2
Where,
N1 = Normality of H2SO4 = 3608
V1 = Volume of H2SO4 = Unknown
N2 = Normality of solution = 0.25 N
V2 = Volume of solution = 70 ml
Putting values in formula N1V1 = N2V2
36.8 V1 = 0.25 × 70 ml
V1 = 0.25 × 70 ml ÷ 36.8 = 0.47 gm
Result:
The solution of H2SO4 is prepared in 70 ml of water, normality of which is 0.25 N.
Prepare 0.25% w/v solution of k2cr2O7

Calculation
Mass of solute = Mass percentage volume of solution
100
0.25×100ml
100
=0.25g
Dissolve 0.25g of k2cr2O7 in a flask of water, making volume 100ml
Result: The 0.25% w/v of k2cr2O7 is prepared which is 0.25g of k2cr2O7 dissolved in 100ml of H2o

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IDENTIFICATION OF PROTEIN (CASEIN)

The Biuret Test

The Ninhydrin Test

Absorbance Assay (280 nm)

Iodine Test for Starch

2. Add about 5 drops of iodine solution to the test tube.

3. Note any colour changes

Emulsion (ethanol) test for Fats

Testing for Nucleic Acids

Procedure 5: The Dische diphenylamine test for DNA

Prepare 0.25% w/v solution of k2cr2O7

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