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Hiper® Protein Estimation Teaching Kit (Quantitative)

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Hiper® Protein Estimation Teaching Kit (Quantitative) U n z i p p i n g G e n e s P r o d u c t I n f o r m a t i o n HiPer® Protein Estimation Teaching Kit (Quantitative) Product Code: HTBC005 Number of experiments that can be performed: 5 Duration of Experiment Protocol Biuret assay: 1 hour Folin – Lowry Assay: 2 hours Bradford assay: 1 hour Storage Instructions: The kit is stable for 12 months from the date of manufacture Store all the kit contents as specified in the brochure 1 Registered Office : Commercial Office 23, Vadhani Industrial Estate,LBS Marg, A-516, Swastik Disha Business Park, Tel: 00-91-22-6147 1919 15 WHO Mumbai - 400 086, India. Via Vadhani Indl. Est., LBS Marg, Fax: 6147 1920, 2500 5764 GMP Tel. : (022) 4017 9797 / 2500 1607 Mumbai - 400 086, India Email : [email protected] CERTIFIED Fax : (022) 2500 2286 Web : www.himedialabs.com The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein Index Sr. No. Contents Page No. 1 Aim 3 2 Introduction 3 3 Materials Required But Not Provided 3 4 Storage 3 5 Biuret Assay 3 Principle Kit contents Important Instructions Procedure Observation and Result Interpretation 6 Folin – Ciocalteau (Lowry) Assay 6 Principle Kit contents Important Instructions Procedure Observation and Result Interpretation 7 Dye Binding (Bradford) Assay 9 Principle Kit contents Important Instructions Procedure Observation and Result Interpretation 8 Troubleshooting Guide 12 2 Aim: To determine the concentration of a protein by three commonly used methods: 1. Biuret Assay 2. Folin – Ciocalteau (Lowry) Assay 3. Dye Binding (Bradford) Assay Introduction: HiPer® Protein Estimation Teaching Kit is designed for rapid and accurate determination of proteins by three most commonly used techniques, each having features that suit it to a particular use. It enables the determination of the concentration of proteins which is frequently required in biochemical work. There is no completely satisfactory single method to determine the concentration of protein in any given sample. Most protein assays take advantage of a reaction between a reagent dye and the protein of interest that will shift or increase the absorbance of a particular wavelength. The choice of the method depends on the nature of the protein, the nature of the other components in the protein sample, desired speed, accuracy and sensitivity of the assay. Materials Required But Not Provided: Glasswares: 1 ml and 10 ml Pipettes, cuvettes, test tubes Reagents: Distilled Water Other requirements: Spectrophotometer/Colorimeter to determine the absorbance in given range, Micropipette and tips, test tube stand Storage: HiPer® Protein Estimation Teaching Kit can be stored at 2-8oC for up to 12 months without showing any reduction in performance. Read Important Instructions before starting the experiment. Store all the reagents as specified in the brochure. 1. Biuret Assay for Protein Estimation: Principle: The Biuret assay is a general protein assay for batches of material for which yield is not a problem. In Biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment containing sodium potassium tartrate. This is known as the biuret reaction because it is chemically similar to a complex that forms with the organic compound biuret (NH2-CO-NH-CO-NH2) and the cupric ion. Biuret, a product of excess urea and heat, reacts with copper to form a light blue tetradentate complex. Fig 1: Biuret Reaction 3 Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue to violet complex that absorbs light at 540 nm. One cupric ion forms a colored coordination complex with four to six nearby peptide bonds. The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction. Thus, the biuret reaction is the basis for a simple and rapid colorimetric reagent of the same name for quantitatively determining total protein concentration. Kit Contents: Table 1: Enlists the materials provided in this kit for Biuret assay with their quantity and recommended storage Quantity Sr. No. Product Code Materials Provided Storage 5 expts 1 TKC226 Protein Standard (50 mg/ml) 4 ml -200C 2 TKC227 Biuret Reagent 110 ml 2-80C 3 TKC228 Protein Sample 1 1.2 ml -200C 4 TKC229 Protein Sample 2 1.2 ml -200C Important Instructions: 1. Read the entire procedure carefully before starting the experiment. 2. All glasswares must be clean and protein free, otherwise it will interfere with the assay. 3. The unknown and standard samples should be treated identically for accurate results. 4. The assay should be carried out at the same time and in the same buffer conditions. 5. Protein test samples (Protein Sample 1 and 2) provided are of different concentrations. Procedure: 1. Take nine tubes and label them as Blank and 1 to 8. 2. Make dilutions of Protein (BSA) standards with concentrations of 1, 2, 4, 6, 8, 10 mg/200 µl by transferring respective amount of BSA from the standard protein solution (50 mg/ml) and adjusting it to a total volume of 200 μl by adding distilled water as mentioned in table 2. 3. Add 2 ml of Biuret reagent to each test tube including the Blank and Unknown tubes. Mix well. 4. Keep at room temperature for 10 minutes. 5. Switch on the Spectrophotometer, select the wavelength at 540 nm and let it warm before taking the absorbance (OD). First take the OD of Blank and make it zero. 6. Remove Blank tube and take the OD of all the tubes and record it. Wash the cuvette after taking OD of each sample. 4 Table 2: Tube No. Blank 1 2 3 4 5 6 7 8 Conc. of BSA Test sample Test Sample 0.0 1 2 4 6 8 10 (mg) 1 2 Amt of Stock 0.0 20 40 80 120 160 200 (μl) Amt of 200 μl 200 μl Distilled Water 200 180 160 120 80 40 0.0 (μl) Amt of Biuret 2 2 2 2 2 2 2 2 2 reagent (ml) Keep at room temperature for 10 minutes Absorbance at 540 nm 7. Plot a Standard Curve of absorbance at 540 nm on “Y” axis versus concentration of protein mg/200 μl on “X” axis. 8. Record the value “x” of Unknown from graph corresponding to the optical density reading of the test sample. Determination of Protein Concentration in Unknown Sample: Protein concentration can be calculated using the following formula: Concentration of Unknown in “mg” Protein Concentration in Test Sample = -------------------------------------------- x 5 Volume of sample in “μl” = ____ mg/ml Observation and Result: 1 2 3 Fig 2: In Biuret Protein Assay, the intensity of the colour increases with increasing protein concentration 5 The absorbance of the protein solution at 540nm increases with increasing protein concentration Interpretation: The Biuret method is carried out by preparing a set of solutions with known protein concentrations and mixing them with the Biuret reagent. A standard curve can be made and the concentrations of unknown protein sample can be derived from the standard curve 2. Folin – Ciocalteau (Lowry) Assay for Protein Estimation: Principle: The Lowry’s method utilizes phenol reagent of Folin and Ciocalteau. This is essentially phosphotungstic phosphomolybdic acid which can be reduced by phenols and many other substances with phenolic rings to ‘molybdenum blue’. Proteins reduce phenol reagent, which may be used therefore for their determination. However, the amount of colour varies greatly with different proteins because it is entirely proportional to their content of tyrosine and tryptophan, other amino acids having little effect. Pretreatment of proteins with alkali and a trace of copper salt greatly increases the colour that is absorbed maximally at 750 nm. Fig 3: Folin Lowry Reaction 6 Kit Contents: Table 3: Enlists the materials provided in this kit forLowry assay with their quantity and recommended storage Quantity Sr. No. Product Code Materials Provided Storage 5 expts 1 TKC230 Protein Standard (1mg/ml) 4 ml -200C 2 TKC231 Folin’s Reagent (2N) 15 ml RT 3 TKC232 Solution A 180 ml RT 4 TKC233 Solution B 2 ml RT 5 TKC329 Solution C 2 ml RT 6 TKC234 Protein Sample 1 1.2 ml -200C 7 TKC235 Protein Sample 2 1.2 ml -200C Important Instructions: 1. Read the entire procedure carefully before starting the experiment. 2. All glasswares must be clean and protein free, otherwise it will interfere with the assay. 3. The unknown and standard samples should be treated identically for accurate results. 4. The assay should be carried out at the same time and in the same buffer conditions. 5. Protein test samples provided are of different concentrations. (Protein Sample 1 and 2) 6. Preparation of 1N Folin’s Reagent (5ml): Dilute Folin’s reagent (2N) with equal amount of Distilled water on the day of use. ( 2.5 ml of Folin’s reagent + 2.5 ml of Distilled water) 7. Preparation of Alkaline Copper Reagent (30 ml): Mix 29.4 ml of Solution A with 0.3 ml of Solution B and 0.3 ml of Solution C on the day of use. Procedure: 1. Take ten tubes and label them as Blank and 1 to 9. Make dilutions of Protein (BSA) standards with concentrations of 10, 20, 40, 80, 120, 160, 200 µ 2. μg/200 l by transferring respective amount of BSA from the standard protein solution (1 mg/ml) and adjusting it to a total volume of 200 μl by adding distilled water as mentioned in table 4.
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