Practical # 1

Scheme for detection of Proteins

General reactions of proteins1. Lead acetate test2. Sulpho salicyclic test3. Esback’s test

Color Reaction of Protein

1. Biuret test2. Xanthoprotiein test3. Millon’s test4. Formaline test5. Sulphur test

Salicyclic acid test

EXPERIMENT OBSERVATION INTEREFERENCE2ml (0.5) + 2 drops of sulpho-salicyclic acid

If ppt dissolve, clear solution

If ppt persists

BJP +ve

Other Proteins present (albumin etc)

Color Reaction of Proteins

Biuret test:-

EXPERIMENT OBSERVATION INTEREFERENCE2ml (5%) NaOH + 2 drops of 1% CuSO4. Divide the solution into 2 parts, T.T.A (test tube A) and T.T.B(test tube B).Add 1ml of distilled water(d/w) in TTAAdd 1 ml of original solution in TTB

Solution containing protein turns purple violet

Peptide linkage protein are present

Coagulation Test:-

EXPERIMENT OBSERVATION INTEREFERENCE3ml (OS) and heat on low flame If coagulation appears Albumin and globulin present

Half Saturation Test

EXPERIMENT OBSERVATION INTEREFERENCETake 3ml of given solution in test tube and add equal amount (3ml) of saturated ammonium(SO4). Now the

If white ppt. appears Globulin is present

solution becomes half saturated

Full Saturation Test

EXPERIMENT OBSERVATION INTEREFERENCETake 5ml of given solution in a test tube and saturate it with solid ammounium sulphate

If white ppt. appears Albumin is present

Precipitation of Protein

Acetic Acid test for Casein

Reagent :- 1% Acetic Acid solution

EXPERIMENT OBSERVATION INTEREFERENCE5ml (OS) + 1% Acetic acid drop by drop till solution changes to white ppt.

White ppt +ve Casein +ve

Principle:- Casein is precipitated out at its iso electric PH(4.6) when 1% Acetic acid is added dropwise.

CT(confirmation test) for Casein(Neuman’s test):-

Reagent:-conc. H2SO4 : Con. HNO3 : Ammonium molybedate solution 5% : Methyl Red Indicator

Principle:- On heating e’ conc. H2SO4 e’ HNO3. Casein is digested and phosphorous is released, NH3 is added to provide alkaline media.

Ammonium molybedate react e’ phosphorous in alkaline media form yellow ppt of phospho-ammonium molybedate

EXPERIMENT OBSERVATION INTEREFERENCEPpt. of acetic acid test + 1 drop of HNO3 + 12 drops of H2SO4, then heat till white fumes appear.Add 1 drop of HNO3, heat till clear solution appear, then cool & dilute e’ 5ml D/wAdd 1 drop of methyl red of strong NH3 till color becomes light yellow + 2ml of ammonium molybedate

Yellow ppt. of phospho-ammonium molybedate appear

Casein Confirmed

Proteinuria:- Pre Renal, Renal (Glomerulus) and Post Renal (bladder trouble, could be stones / trauma)