Isolation of Casein

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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; condensation...
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