Proteins

Introduction
Proteins are very large molecules with molecular weights that range from 6000 to millions of grams per mole. While proteins can be used as an energy source by the body, this is not their primary role. Proteins are essential for the catalysis of most of the body's chemical reactions, for structural support and for the transport/storage of vital nutrients. As proteins and amino acids (the components of proteins) are not stored in the body, some protein intake is required each day.

I- General properties of amino acids

Amino acids are the building blocks of proteins. In humans, the amino acids used are ! amino acids, which means the carboxylic acid group and the amino group are located on the same carbon. The -amino and -carboxyl groups on amino acids act as acidbase groups, donating or accepting a proton as the pH is altered. At low pH, both groups are fully protonated, but as the pH is increased first the carboxyl group and then the amino group loses a hydrogen ion. For the standard 20 amino acids, the pK is in the range 1.82.9 for the -carboxyl group and 8.810.8 for the -amino group. Those amino acids with an ionizable side-chain have an additional acidbase group with a distinctive pK.

Isoelectric Point (pI): is the pH at which the amino acid has no net charge. The isoelectric point of an amino acid is the average of the pKa values of the protonation transitions on either side of the isoelectric species. For example, aspartic acid isoelectric species exists in the pH domain between pKa1 (2.1) and pKa2 (3.9). Thus, the pI of aspartic acid is 3.0. Reactive groups in amino acids include -NH2 and -COOH groups and groups present on side R chains. In peptides and proteins only the side chain is available for reactions besides amino and carboxylic groups at the terminal ends. When amino acids are linked together, amide or peptide bonds are formed. The formation of an amide group is shown in the reaction below, in which two amino acids react to form a dipeptide.
- H2O

Reactions of this type can continue to link many amino acids together to form polypeptides. When the number of amino acids in the molecule reaches about 50, it is considered a protein. Proteins can be classified as simple or complex. A simple protein is composed only of amino acids. Complex proteins, which are far more common, incorporate other non-amino acid groups in their structure.

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Exp. # 1 The solubility of amino acids test

Principle: In general amino acids are soluble in aqueous solution and the lowest solubility around their isoelectric point (pI), but slightly soluble or insoluble in organic solvents. Nearly all aliphatic amino acids exist as zwitterions (The doubly charged situation of the amino acid). The strong positive charge on the NH3+ group induces a tendency for COOH group to loose a proton, so that amino acids are stronger acids than many of the weak acids. For example glycine has a pKa of 2.4 in comparison to Acetic acid 4.8. Materials and reagents: 1. Hydrochloric acid (0.1mol/l) 2. Sodium hydroxide (0.1mol/l) 3. Ethanol 4. Chloroform 5. Amino acids (glycine, glutamic acid, lysine, and alanine) Method: Examine the solubility of the above amino acids in water, dilute acid, and dilute alkali, ethanol, and chloroform. 1) Add 1 2 ml of water, dilute acid, and dilute alkali, ethanol, and chloroform solution to different labeled tubes. 2) Add a very small amount of the solid amino acids. 3) Examine the solubility of the above amino acids. *** How do you interpret the results?

Exp. #2 The ninhydrin test

Principle: Ninhydrin (triketohydrindene hydrate) is a powerful oxidating agent which leads to the oxidative deamination of alpha-amino (NH2) groups between pH 4 and pH 8. It is very important for the detection and the quantitative analysis of amino acids. Ninhydrin also reacts with primary amines and ammonia however the formation of carbon dioxide is quite diagnostic for amino acids. Alpha amino acids yield a purple substance that absorbs maximally at 570 nm.

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Imino acids (Proline and hydroxyproline) yield a yellow product (absorption maximum 440 nm). Ninhydrin reaction is very sensitive and is ideal for the detection of amino acids on chromatograms and their quantitative determination.

Ninhydrin reaction is very sensitive and is ideal for the detection of amino acids on chromatograms and their quantitative determination in column fractions. Materials and reagents: 1. Amino acids(1g/lglycine, tyrosine, and tryptophan) 2. Ninhydrin (2g/l prepare fresh) Method: 1- Place 1 ml of the amino acid solution in a test tube. 2- Adjust the pH to about neutrality. 3- Add five drops of Ninhydrin solution. 4- Boil for 2min. Optional: Determine the limits of sensitivity of the reaction by carrying out the test on serial dilutions of glycine until a negative result is obtained. Exp. #3 The xanthoproteic reaction. Principle: Xanthoproteic comes from the Greek word xanthos which means yellow. Boiling concentrated nitric acid reacts with tyrosine, tryptophan and phenylalanine (phenylalanine give weakly positive reaction as it contain an inactive benzene ring) to yield yellow products. The intensity of yellow color deepens upon the addition of alkaline solution that form orange colored salt in the basic medium.

Materials and reagents: 1- Amino acids (1g/l glycine, tyrosine, tryptophan and phenylalanine). 2- Phenol (1 g/l).
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3- Nitric acid (conc.) 4- Sodium hydroxide (10 mol/l) Method: 1- Add an equal volume of conc. Nitric acid to about 0.5 ml of the amino acid solution. 2- Cool, and observe the color change. 3- Add sufficient NaOH to make the solution strongly alkaline. ^-^-^- Yellow color in acid solution which turns deeper yellow then bright orange with alkali constitutes a positive result. 4- Repeat the test with the phenol solution. Phenylalanine gives a negative or weakly positive solution.

Exp. # 4 Millons reaction

Principle: Compounds containing the hydroxybenzene radical react with millons reagent to form red complexes. The only phenolic amino acids are tyrosine and its derivatives and only these amino acids give a positive reaction. The original Millons reagent was a solution of mercuric nitrate in 50% v/v nitric acid, but modifications are now used that are less liable to interference from organic salts. Materials and reagents: 1- Amino acids (1 g/l glycine, tyrosine, and phenylalanine). 2- Millons reagent (150 g/l solution of mercuric sulphate in 15% v/v sulphuric acid). 3- Phenol (1 g/l). 4- Sodium nitrate (10 g/l). 5- Boiling water bath. Method: 1- Add five drops of Millons reagent to 1 ml of the test solution. 2- Heat in a boiling water bath for 10 min. Then cool to room temperature. 3- Add five drops of sodium nitrate solution; a positive result is indicated by a brick red color.

Exp. # 5 Glyoxylic reaction for tryptophan (Hopkins-Cole test)

Principle: The indole group of tryptophan reacts with glyoxylic acid in the presence of conc. Sulphuric acid to give a purple color. Glacial acetic acid that has been exposed to the light contains glyoxilic acid. Also protein can be detected by this test as concentrated H2SO4 at the solution interface hydrolyses protein to form free tryptophan, which reacts with glyoxylic acid to form violet-purple complex.

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Materials and reagents: 1. Amino acid (1g/l glycine, tyrosine, and tryptophan). 2. Glyoxylic acid; Glacial acetic acid which has been exposed to light. 3. Sulphuric acid (conc.). Method: 1- Add 2ml of glacial acetic acid to 2ml of the test solution. 2- Pour about 2 ml of conc. H2SO4 carefully down the sides of a sloping test tube so as to form two layers. 3- Observe any color change at the liquid junction. - - - A positive result is shown by the formation of a purple-violet ring at the junction of the two liquids.

Exp. # 6 Paulys reaction

Principle: The basic principle involved in pauly's test is diazotization. The sulphanilic acid gets diazotised in the presence of sodium nitrite and sodium carbonate with the sample. Diazotized sulphanilic acid reacts with the phenol group of tyrosine, and imidazole ring of histidine to form highly-colored azo compounds. This test reacts with histidine to give a red color and with tyrosine to give an orange color. Tryptophan gives yellow color. The diazonium compounds are only formed in the cold, so all solutions are cooled in ice before diazotization. Materials and reagents: 1. Amino acids (1g/l glycine, tyrosine, histidine, and tryptophan). 2. Sulphanilic acid (10g/l solution in 1mol/l Hydrochloric acid) 3. Sodium nitrate (50g/l) 4. Sodium carbonate (10g/l) Method: 1- Add 2 ml of the test solution to test tube, cool in ice. 2- Mix 1 ml of sulphanilic acid with the test solution, keep on ice. 3- Add 1ml of NaNO2 solution and leave in the cold for 3min. 4- Make the solution alkaline by the addition of 2 ml of Na2CO3 and note the colors formed.

Exp. #7 Ehrlichs reagent

Principle: Ehrlich's reacts with a number of organic compounds such as indoles, aromatic amines, and ureides to give colored copmounds. Ehrlich's reagent is well known strong electrophile which reacts with the electron-rich !-carbon of indole rings to form a blue-coloured compounds. The reason it is used as the confirmative test for tryptophan utilization by some bacterial species by Indole test. Also, biologically, Ehrlich's reagent used for detection and content determination of Urobilinogen in urine samples as it give dark pink or red color. In industrial chemistry it is used for hydrazine (N2H4) spectrophotometeric determination at 458 nm as Ehrlich's reagent form distinct yellow color with hydrazine. Materials and reagents: 1. Ehrlichs reagent (100g/l p-dimethylaminobenzaldehyde in conc. Hydrochloric acid) 2. Amino acids (1g/l glycine, tryptophan, and hydroxyproline).
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3. Urea (1g/l). Method: 1- Add 2 ml of Ehrlichs reagent to 0.5 ml of the test solution. 2- Observe the color.

Exp. # 8 The nitroprusside test

Principle: Thiol groups react with sodium nitroprusside (Na2Fe(CN)5 NO) in the presence of excess ammonia to give a red color. This test is specific for cysteine.

Materials and reagents: 1. Sulphur amino acids (1 g/l cysteine, cystine, and methionine). 2. Sodium nitroprusside (20 g/l prepare fresh). 3. Ammonium hydroxide. Method: 1. Mix 0.5 ml of a fresh solution of sodium nitroprusside with 2ml of the test solution. 2. Add 0.5 ml of ammonium hydroxide. 3. Observe the color. Exp. # 9 The Sakaguchi reaction Principle: The only amino acid containing the guanidine group is arginine, and this reacts with ! naphthol and an oxidizing agent bromide water to give a red color. Materials and reagents: 1- Amino acids (1 g/l glycine and arginine). 2- Guanidine (1 g/l glycocyamine, methylguanidine and creatine). 3- Urea (1 g/l). 4- Sodium hydroxide(10mol/l). 5- ! Naphthol(10g/l in alcohol). 6- Bromine water (add a few drops of bromine to 100 ml of water and shake; do this in a fume chamber). Caution: Bromine gives very nasty burns if spilt on the skin. Method: 1- Mix 1 ml of strong alkali with 3 ml of the amino acid solution. 2- Add two drops of ! naphthol. 3- Mix thoroughly and add four or five drops of bromine water. 4- Note the color formed.

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II- General reactions for proteins

Albumins Albumin (Latin: albus, white) refers generally to any protein readily soluble in water, dilute acids and alkalies which may be precipitated out from solution using high salt concentration in a process called 'salting out'. Albumin experiences heat coagulation (protein denaturation). Substances containing albumin, such as egg white, are called albuminoids. Albumin is the main protein of plasma; it binds water, cations (such as Ca2+, Na+ and K+), fatty acids, hormones, bilirubin and drugs - its main function is to regulate the colloidal osmotic pressure of blood. Globulins Globulins are insoluble or sparingly soluble in water, but their solubility is greatly increased by the addition of neutral salts such as sodium chloride in a process called 'salting in'. These proteins are coagulated by heat. They are deficient in methionine. Example - Serum globulin, Fibrinogen, Myosin of muscle Casein Casein (from Latin "cheese") is the predominant phosphoprotein that accounts for nearly 20% of proteins in cow milk and cheese. Casein is not coagulated by heat. It is precipitated by acids and by rennet enzymes. Casein consists of a fairly high number of proline peptides, which do not interact. There are also no disulfide bridges. As a result, it has relatively little tertiary structure. Because of this, it cannot denature. It is relatively hydrophobic, making it poorly soluble in water. The purified protein is water insoluble. While it is also insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in diluted salt solutions such as sodium oxalate and sodium acetate. Gelatin Gelatin is produced by partial hydrolysis of collagen extracted from the boiled bones, connective tissues, organs and some intestines of animals. The natural molecular bonds between individual collagen strands are broken down into a form that rearranges more easily. Gelatin melts to a liquid when heated and solidifies when cooled again. Gelatin is slightly soluble in cold water that disperse more easily in the acidic solution. Gelatin is also soluble in most polar solvents Although gelatin is 98-99% protein by dry weight, it has less nutritional value than many other protein sources. Gelatin is unusually high in the non-essential amino acids glycine and proline (i.e., those produced by the human body), while lacking certain essential amino acids (i.e., those not produced by the human body). It contains no tryptophan and is deficient in isoleucine, threonine and methionine. Peptones (Peptides) Peptones (from the Greek "to digest") are short polymers of amino acids. They have the same chemical structure as proteins, but are shorter in length. One convention is that peptide chains that are short enough to be made synthetically from the constituent amino acids are called peptides rather than proteins. Peptones are derived from animal milk or meat digested by proteolytic digestion. In addition to containing small peptides, the resulting spray-dried material includes fats, metals, salts, vitamins and many other biological compounds. Peptone is used in nutrient media for
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growing bacteria and fungi. Glutathione Glutathione is a tripeptide thiol composed of glutamic acid, cysteine, and glycine. The sulfhydryl (thiol) group (SH) of cysteine serves as a proton donor and is responsible for the biological activity of glutathione as a scavenger for reactive oxygen species (ROS) such as free radicals and peroxides. Glutathione has been called the "master antioxidant", moreover, it regulates the actions of lesser antioxidants such as vitamin C, and vitamin E within the body. A deficiency of glutathione can cause hemolysis and oxidative stress.

Exp. # 10 The Biuret test for peptide bonds

Principle: The name of the test comes from the compound biuret, which gives a typically positive reaction. Biuret is obtained by heating urea to about 180 C. In a kind of chelation reaction a blue color is formed with Cu2+.

In a similar chelation reaction Cu2+ reacts with protein to form Cu2+-protein complex.

In this experiment, alkaline copper sulphate reacts with compounds containing two or more peptide bonds to give the violet-colored complex. The depth of the color obtained is a measure of the number of peptide bonds present in the protein. The reaction is not absolutely specific for peptide bonds, since the compounds containing two carbonyl groups linked through hydrogen or carbon atom will give a positive result. Materials and reagents: 1. Copper sulphate (10g/l of CuSO4.5H2O). 250 ml 2. Sodium hydroxide (10mol/l). 2l 3. Proteins (5g/l albumin, casein, gelatin, and Peptone: casein is dissolved a little dilute NaOH and the other proteins in saline). 500 ml
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4.

Glutathione (5g/l).

500 ml

Method: 1- Add five drops of copper sulphate solution to 2 ml of the test solution. 2- Add 2 ml of NaOH; mix thoroughly. 3- Note the produced colors.

Exp. # 11 Denaturation by heat and extreme pH.

Materials and reagents: 1. Proteins as in exp.10 2. Hydrochloric acid (1mol/l) 3. Sodium hydroxide (1mol/l) 4. Nitric acid (conc.) 5. Boiling water bath Method: 1- Place 5 ml of each protein in three test tubes. 2- Add 0.5 ml of HCL, 0.5ml of NaOH, and 0.5ml of water to these 3 tubes of each protein. 3- Place the tubes in a boiling water bath for 10 min then cool to room temperature. 4- Adjust the acid and alkaline tubes to neutrality. *** Comment on your observations. 5- Slowly pour 2 ml of conc. HNO3 down the sides of test tubes containing 2 ml of protein solution so as to form two layers. 6- Carefully mix the two liquids. *** Record your observations.

Exp. # 12 Precipitation by heavy metals.

Principle: At pH7 and above, proteins are usually negatively charged so that addition of a positively charged metal ion neutralizes this charge and the proteins come out of solution. Precipitation by heavy metals is therefore, most effective at neutral to slightly alkaline pH values, although the solution must not be too alkaline otherwise there is a risk of metal hydroxides being precipitated. The formed precipitate is frequently soluble in excess of the heavy metal ion solution since the excess ions confer a stabilizing positive charge on the particles. Materials and reagents: 1. Proteins as in exp.10 . 2. Heavy metals (0.1 mol/l copper sulphate, lead acetate and mercuric nitrate) Method: 1- Add a few drops of the heavy metal solution to 2 ml of the protein solution. 2- Record your observations. *** What happens when excess reagent is added?

Exp. # 13 Precipitation by acidic reagent.

Principle: These compounds are acids and carry a large negative charge which neutralizes a positively charged protein to form an insoluble salt. The acidic reagents are, therefore, most
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effective at acidic pH values where proteins are positively charged. Materials and reagents: 5- Proteins as in exp.10 . 6- Acidic reagents (20% w/v sulphosalicylic acid, saturated picric acid, 10% w/v tannic acid, and 20% w/v trichloracetic acid). Method: 1- Add five drops of the acidic reagent to 1-2 ml of the protein solution. *** What is the effect of adding an excess of the reagent? 2- Slowly add dilute NaOH. 3- Observe the result as the pH decreases.

III- Colorimetric Methods for Quantitative Determination of Proteins.

In general, there is no completely satisfactory or best way to determine the concentration of protein in a given sample. The choice of the method depends on: 1) The nature of the protein under test, 2) The nature of the components in the protein sample (i.e. contaminants), 3) The desired speed, 4) Accuracy, and 5) Sensitivity of assay. Moreover, such assays needed to be 6) Reproducible, 7) Simple and quick to perform, and 8) Inexpensive. There are two general classes for the most commonly used colorimetric assay methods to determine protein concentration; B) Cu redox based chemistry as in 1) Biuret, 2) Lowry and 3) BCA assay methods. C) Coomassie blue binding as in Bradford assay method. - Protein denaturation in these methods is required in order to get maximal color change. Denaturation in Biuret, Lowry, and BCA due to high NaOH concentration. Denaturation in Bradford due to high H3PO4 concentration. In addition to the common ways to express concentration by Molarity: mol/l, % solution: 1% solution = (1 g/100 ml); the above mentioned assay methods usually expressed in amount/vol: mg/ml and sometimes in mg/dl that express the total protein content. Moreover, proteins can be expressed by another unit; Enzyme activity: mol product/(min ml E). Enzyme activity means the amount of enzyme needed to convert 1 mol substrate to product in 1 min = 1 EU or U (Enzyme Unit). General Guidelines for all Colorimetric Methods: 1. Preparation of range of protein standard concentrations (usually BSA). 2. Addition of fixed volume of standard to fixed volume of dye solution. 3. Incubation for specific time and specific temperature. 4. Measure of absorbance at single wavelength specific to method (i.e. specific to dye color). 5. Plotting absorbance vs. BSA standard concentration (mg/ml) to generate the standard curve. 6. Repeat step 2 - 4 for the unknown protein.
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7. Application of unknown absorbance reading to the standard curve to find the working unknown concentration. Finally, multiply the working unknown concentration with dilution factor to find the original unknown concentration.

A- Biuret Method (540 nm)

Introduction:
The Grandfather of all colorimetric methods. Principle: Two reactions are involved: (1) Chelation reaction in basic medium:

(2) Redox reaction: Cu2+ + (Tyr, Trp, polar AAs)red ! Cu+ + (AAs)ox Is the basis for the Lowry and BCA methods. Least sensitive (100 1000 fold less sensitive than Lowry, BCA, or Bradford). Disadvantages: 1) Requires relatively large amounts of protein (1-20 mg). 2) Not suitable in the presence of ammonium salts. Lipoid materials may yield a cloudy reaction mixture which can be cleared by shaking with 1.5 ml of diethyl or petroleum ether and then centrifuge and read the aqueous phase. Bile pigments absorb light very weakly in the region of 540 560 nm.

Materials and reagents:

A. Biuret reagent: 1. 1.5 g CuSO4.5H2O 2. 6 g sodium potassium tartrate (NaKC4H4O6.4H2O)
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- Dissolve each compound separately in sufficient distilled water, add (1) to (2). 3. 300 ml 10 % NaOH -Add solution from (3) with constant swirling to the solution of (1+2) 4. 1 g KI. 5. Dilute to 1 liter with distilled water. Store in paraffin-lined bottle. Note: This reagent can be kept indefinitely but must be discarded if, as a result of contamination or faulty preparation, it show signs of depositing any black or reddish precipitate. Biuret reagent may be prepared without KI, however, the addition of 0.1 % KI may prevent excessive reduction and has not detectable effect on the rate, degree, or quality of biuret color. B. Standard protein solution. Bovine serum albumin (BSA): 10mg/ml in distilled water. C. Unknown protein sample. 4ml, it will be given by your assistant.

Procedure:
1. 2. 3. 4. Prepare several dilutions of standard (BSA) solution with water to a final volume of 1.0 ml as shown in table (1) together with a blank tube containing all reagents except the protein. Dilute the unknown protein solution to 1 ml as in table 1. Mix solutions thoroughly. Add 4 ml of Biuret reagent, mix the solutions thoroughly. Allow the samples to stand for 30 minutes at room temperature OR place all the test tubes, and contents into a water bath at 50C for 10 minutes (The color is stable for about 60 min at room temperature). Measure the intensity of the color in each tube at 540 nm against blank which is set at zero absorption. data and report the protein concentration of the unknown sample. A typical Biuret calibration curve is shown in next figure.

5.

6. Prepare the standard protein calibration curve (OD 540 versus mg/ml protein) from these

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Table 1: Preparation of Blank, Standard and Unknown samples for the Biuret assay. Tube Protein H2O (ml) Biuret O.D No. solution (ml) reagent (ml) 540 Blank Blank 1 0.0 1.0 4.0 Blank 2 0.0 1.0 4.0 Standard Std. 3 0.1 0.9 4.0 protein Std. 4 0.2 0.8 4.0 BSA Std. 5 0.5 0.5 4.0 (10 mg/ml) Std. 6 0.8 0.2 4.0 Std. 7 1.0 0.0 4.0 Unknown Ukn. 8 0.1 0.9 4.0 protein Ukn. 9 0.5 0.5 4.0 Ukn. 10 1.0 0.0 4.0 Note: Duplicates can be used to reduce any error that may be generated during preparation.

B- Lowry Method: (660 nm)

Introduction:
Principle: The final deep blue color formed by the Follin- Lowry reagent is due to: 1) Reaction of proteins with cupric ions in alkaline solution as in Biuret assay. 2) Redox reaction of Biuret (Generation of Cu+) involved in a second redox reaction which is responsible for the enhanced color change: (i) Cu2+ + (Tyr,Trp, polar AAs)red ! Cu+ + (AAs)ox (ii) Cu+ + Folinsox ! Cu2+ + Folinsred (Dark blue) due to Folins reagent contain phosphomolybdic phosphotungtic acid. Therefore, the intensity of the color depends mainly on the amount of Tyr and Trp amino acids present in the protein. More sensitive (About 100 times) than Biuret assay in which detection may reach 5 g/ml. Disadvantages: - Sensitive to variety of contaminants. - Non-linear concentration dependence. Therefore, standard curve must be generated each time. - Correct mixing (there are two reagents) and timing are critical. Lowry method is very good for proteins containing chromophores such as hemes and flavins.

Materials and reagents:

A. Alkaline copper (Reagent B) reagent: Reagent I : 2 g CuSO4.5H2O. Reagent II: 2 g sodium potassium tartrate or sodium or potassium tartrate. Reagent A: 2 g Na2CO3 in 0.1ml NaOH. ! Reagent B: Mix in order; 0.1 ml reagent I, 0.1ml reagent II, 10ml reagent A. Prepare fresh before use. B. Follin-Phenol reagent (2 N solution): Dilute Follin-Phenol reagent 1:1 with distilled water, make just before use. Keep in dark. C. Bovine serum albumin (BSA) standards: 1- 0.02 mg/ml. 2- 0.05 mg/ml. 3- 0.10 mg/ml.
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4- 0.15 mg/ml. 5- 0.20 mg/ml Standards are prepared in distilled water and can be stored in refrigerator but they should be discarded if bacterial growth is evident or if a change in a standard curve is not attributable to reagent. D. Unknown protein sample: Dilute the unknown protein sample with distilled water as in Table 2. If the sample to be analyzed contains ammonium sulfate, dilute with 10% Na2CO3. If ammonium sulfate concentration is greater than 0.25% after all reagents have been added, readings will be low.

Procedure: 1- Take 0.5 ml aliquots of BSA standard in small slender test tubes for a standard 234567calibration curve and 0.5 ml distilled water for reagent blank as shown in Table 2. Take 0.5 ml, 0.25 ml and 0.1ml aliquots of unknown sample to test tubes. Make up volume of 0.25 ml aliquots and 0.1 ml aliquots to 0.5 ml with distilled water (Table 2) Add 2.5 ml of reagent B (alkaline copper reagent) into the test tubes containing 0.5 ml of the sample or standard solutions. Mix by vortex and allow standing undisturbed for 10 minutes. Add 0.25 ml in Follin-phenol reagent rapidly. Mix immediately within 2 seconds by vortex before the addition of Follin-phenol reagent to the next tube. Allow the samples to stand for 30 minutes at room temperature OR place all the test tubes, and contents into a water bath at 50C for 10 minutes (The color is stable for at least two hours at room temperature). Cool rapidly in a beaker of tab water. Measure the intensity of color in each tube at 660 nm against Blank which is set at zero absorbance. Plot the standard calibration curve from the corresponding absorbance values of BSA standards (OD 660 versus mg protein). Calculate protein concentrations of the dilute sample solution by using the standard curve. Multiply by the dilution factor for protein concentrations of the given sample.

Table 2: Preparation of Blank, Standard and Unknown samples for Lowry assay. Tube Protein H2O, Reagent Follin O.D No. solution, ml ml B, ml Phenol, ml 660 nm Blank Blank 1 0.0 0.5 2.5 0.25 Blank 2 0.0 0.5 2.5 0.25 0.05 mg/ml BSA std. 3 0.5 2.5 0.25 0.1 mg/ml BSA std. 4 0.5 2.5 0.25 0.15 mg/ml BSA std. 5 0.5 2.5 0.25 0.2 mg/ml BSA std. 6 0.5 2.5 0.25 Unknown Ukn. 8 0.1 0.4 2.5 0.25 protein Ukn. 9 0.25 0.25 2.5 0.25 Ukn. 10 0.5 2.5 0.25 Note: Duplicates can be used to reduce any error that may be generated during preparation.
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C- BCA Method (560 nm):

BCA = BiCinchoninic Acid. Same redox reaction of Biuret that generates Cu+. Cu+ forms coordination complex with BCA reagent. Markedly influenced by protein-to-protein variation.

D- Bradford Method (595 nm):

Recently this method is the most commonly used protein colorimetric assay. Principle: - Coomassie blue + protein ! Blue complex Advantages: - Simple one-step method with 5 minute incubation time at Room Temp. - Sensitive and accurate. Disadvantages: - Non-linear concentration dependence. - Stains glassware and glass cuvettes (Disposable plastics can solve this point). - Interfered by detergents.

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