Isolation of Casein
Experiment 1: Isolation of Casein from Milk * pH of milk – 6.6 * milk = 87.1% water, 4.9 % CHO 3.9 %, fats, 0.7% minerals
Experiment 2: Protein Hydrolysis and Characterization | Reagents | Principle | Test for | Positive Result | Negative Result | Biuret | CuSO4, NaOH | Complexation of Cu+2 with amide N atoms | Polypeptide bonds | Violet/purple solution | Blue color solution | Sakaguchi | 10% NaOH, 0.02% α-naphtol solution, 2% NaOBr | arginine condenses with α-naphtol and NaOH | Guanido group (arginine) | orange solution | light yellow solution | Ninhydrin | 0.1% ninhydrin solution (1,2,3-indanetrione monohydrate / triketohydrindene hydrate) | Oxidative deamination and decarboxylation; reduction of ninhydrin | free α-amino and carboxyl groups | purple-blue color solution | Yellow solution | Xanthoproteic | Conc. HNO3, 50% NaOH | Nitration substitution in Benzene ring | aromatic amino acids | yellow precipitate then turns orange when neutralized with NaOH | clear solution, no change in color | Hopkins-Cole | Hopkins-Cole reagent | Condensation of indole group with glyoxylic acid and H2SO4 | tryptophan | Violet ring at the junction where the two layers meet | Absence of purple ring | * Acid hydrolysis is a better procedure than Alkaline hydrolysis because of the less destruction of amino acids
Experiment 3: Protein Assay by Bradford Method * Coomassie Brilliant Blue G-250 * Bradford Reagent * Bovine Serum Albumin = BSA * Reagent blank = test tube #1 = 0mL BSA stock solution, 1mL distilled H2O * 595nm * Plot A595 vs concentration of BSA
Experiment 4: Enzymatic Activity of Salivary Amylase * Iodine test – presence of starch * 1/t vs temperature/pH
Experiment 5: General and Specific Tests for Carbohydrates | Reagents | Principle | Test for | Positive Result | Negative Result | Molisch | Molisch Reagent ,Conc. H2SO4 | hydrolysis, dehydration forming either a furfural or a 5-hydroxufurfural;
Experiment 2: Protein Hydrolysis and Characterization | Reagents | Principle | Test for | Positive Result | Negative Result | Biuret | CuSO4, NaOH | Complexation of Cu+2 with amide N atoms | Polypeptide bonds | Violet/purple solution | Blue color solution | Sakaguchi | 10% NaOH, 0.02% α-naphtol solution, 2% NaOBr | arginine condenses with α-naphtol and NaOH | Guanido group (arginine) | orange solution | light yellow solution | Ninhydrin | 0.1% ninhydrin solution (1,2,3-indanetrione monohydrate / triketohydrindene hydrate) | Oxidative deamination and decarboxylation; reduction of ninhydrin | free α-amino and carboxyl groups | purple-blue color solution | Yellow solution | Xanthoproteic | Conc. HNO3, 50% NaOH | Nitration substitution in Benzene ring | aromatic amino acids | yellow precipitate then turns orange when neutralized with NaOH | clear solution, no change in color | Hopkins-Cole | Hopkins-Cole reagent | Condensation of indole group with glyoxylic acid and H2SO4 | tryptophan | Violet ring at the junction where the two layers meet | Absence of purple ring | * Acid hydrolysis is a better procedure than Alkaline hydrolysis because of the less destruction of amino acids
Experiment 3: Protein Assay by Bradford Method * Coomassie Brilliant Blue G-250 * Bradford Reagent * Bovine Serum Albumin = BSA * Reagent blank = test tube #1 = 0mL BSA stock solution, 1mL distilled H2O * 595nm * Plot A595 vs concentration of BSA
Experiment 4: Enzymatic Activity of Salivary Amylase * Iodine test – presence of starch * 1/t vs temperature/pH
Experiment 5: General and Specific Tests for Carbohydrates | Reagents | Principle | Test for | Positive Result | Negative Result | Molisch | Molisch Reagent ,Conc. H2SO4 | hydrolysis, dehydration forming either a furfural or a 5-hydroxufurfural;