1. Urine Analysis Protein

2. Protein: Urine normally contains a scant amount of protein, which derives both blood and urinary tract itself. Proteins present in urine include albumin, micro globulin, immunoglobulin light chains and enzymes. Proteinuria may be detected in patients with damage in the glamorous or damage in the renal tubles.When the glomerular membrane is damaged greater amount of albumin pass into the glomerular filtrate. Myoglobinemia occurs due to extensive destruction of muscle fibers and it will lead to excretion of Myioglobin in the urine. Multiple myeloma a neoplastic disorder of plasma cells is another example of a systematic disorder which may cause proteinuria. Estimation Of Proteins Reagents A.3% Acetic Acid: 3ml of glacial acetic acid is dissolved in 97 ml of distilled water. Procedure: The urine specimen is centrifuged and the supernatant is collected. Test tube is heated in a Bunsen burner and look for cloudiness at the top. If no cloudiness appears, test tube is removed and 2-3 drops of 3% acetic acid are added. The tube is heated again and watched for cloudiness.If cloudiness disappears, report as negative. If it persists report as positive. B. Sulphosalicylic Acid Test Reagent: 500g of sulphosalicylic acid is poured in 100ml flask and dissolve to 100ml with distilled water. Procedure: 5ml of the reagent is taken and 0.5ml of the urine specimen is added to it and watch for turbidity.

3. ESTIMATION OF SERUM PROTEIN Principle: Proteins and peptides react with alkaline copper tartarate to give a violet colored complex, which is measured calorimetrically at 450 nm.The intensity of the color is directly proportional to the protein concentraction.The name biuret comes from analogous reaction that takes place between the cupric ion and the organic compound biuret (an ammonia compound). Specimen preferred: Serum Reagent required: Biuret reagent 1: a) Dissolve 5g of potassium iodide in 0.25N sodium hydroxide, i.e,10g of NaoH in 1 litre of carbonate free water. b) Dissolve 15g of copper sulphate in 70-80 ml of distilled water. c) Prepare a solution of 45 g of Rochelle salt (potassium sodium tartarate tetra hydrate ) in 600 ml of biuret diluent No.1 d) Stock biuret: add slowly the copper sulphate solution to the sodium tartarate solution. Mixing must be done in order to prevent the reduction of cupric ions by tartarate. Working biuret solution: Take 20ml of stock solution and dilute to 100 ml with the biuret diluents. Reagent II: Saline solution : Dissolve 8.5g of Nacl in 1000 ml of water. Reagent III: Standard protein solution: It is available commercially. Procedure: Three test tubes(clean) are taken and marked as blank, test and standard.1 ml of Biuret reagent is added in all the tubes. To it 20µL of standard, distilled water, and test samples are added

4. respectively. The tubes are incubated at room temperature for 15-30 minutes. The optical density is measured at 540 nm. Normal value: 6-8g/dl Calculation = OD of test-OD of the blank _________________________ ˣ concentration of standard OD of the standard-OD of blank ESTIMATION OF SERUM ALBUMIN Method: Bromocresol green dye method Principle: Albumin binds with bromocresol green in a suitable buffer, making a shift in the dyes peak absorption wave length which is read at 630 nm and the colour developed is directly proportional to the albumin concentration. Specimen Preferred: Serum Reagents required:

5. 1.Succinic acid : Dissolve 5g of succinic acid in 80 ml of distilled water and make upto 100ml. 2.Brig-35: Dissolve 30g of Brig-35 in about 30ml of hot distilled water.Transfer to 100 ml flask and make up the volume with distilled water. 3.NaoH: Dissolve 10g of NaoH in 500ml of fresh distilled water, transfer to 1 litre flask and make up the volume. 4.Dye Solution: Take 100 mg of sodium azide in 1 liter beaker, add 99ml of distilled water and 4 ml of Brig-35(soln.2), stir until dissolved. Adjust the PH to 4.2 to 0.05 (Isoelecteric PH for protein to bind-where maximum affinity could be observed). 5.Albumin standard : Commercially available or prepare by dissolving 10g of dry albumin powder to 80 ml of Nacl and sodium azide solution. Procedure: 1 ml of the reagent (Bromocresol) is added in 3 test tubes marked as Blank, Test and standard. To it, 1 ml of deionized water is added to all the tubes.20µL of dis.water,test sample and standard are added respectively. The tubes are incubate for 10 minutes at room temperature and O.D is read at 630 nm. Calculation: OD of Test-OD of blank ______________________ Concentration of standard OD of standard –OD of blank Globulin = Total protein-albumin Normal Value: albumin; 3.5-5g/dl Globulin: 2.3-3.5 g/dl