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Questions and Answers
What is the primary purpose of Lowry's method?
Which reagents are involved in the color development during protein estimation using Lowry's method?
What is the significance of using the blank tube in the Lowry's method?
What is an essential step before mixing reagents in Lowry's protein estimation method?
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Which component of the protein reacts with the Folin-Ciocalteau reagent in Lowry's method?
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What happens during the preparation of the Folin-Ciocalteau reagent?
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In the context of Lowry's method, what is meant by hydrolyzing proteins?
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Which of the following describes the role of alkaline copper solution in Lowry's method?
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Study Notes
Protein Estimation by Lowry's Method
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Principle: The Lowry's method quantifies protein by measuring the color developed from two reactions:
- Biuret reaction: A reaction between the protein and alkaline cupric tartrate
- Reduction of Folin-Ciocalteau reagent: Reduction of the reagent by the amino acids tyrosine and tryptophan in the protein.
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Materials:
- Reagent A: 2% Sodium Carbonate in 0.1N Sodium Hydroxide
- Reagent B: 0.5% Copper Sulphate in 1% potassium sodium tartrate
- Reagent C: Alkaline Copper solution (50mL of Reagent A mixed with 1mL of Reagent B)
- Reagent D: Folin-Ciocalteau Reagent (prepared by refluxing a mixture of sodium tungstate, sodium molybdate, water, phosphoric acid, and hydrochloric acid with lithium sulfate and bromine water)
- Protein solutions: A stock standard and a working standard
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Procedure:
- Protein extraction: Extract protein from the sample using a buffer suitable for the enzyme assay. Grind 500mg of the sample with the buffer, centrifuge, and use the supernatant for protein estimation.
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Estimation:
- Set up a series of test tubes containing varying volumes of the working standard (0.2, 0.4, 0.6, 0.8, and 1mL).
- Add known volumes of the sample extract to other test tubes (0.1mL and 0.2mL).
- Bring the volume in all test tubes to 1mL with water, and include a blank with only water.
- Add 5mL of Reagent C to each tube, mix well, and let stand for 10 minutes.
- Add 0.5mL of Reagent D to each tube, mix well, and let stand for 30 minutes.
- Measure the absorbance at 750nm, using the blank to zero the spectrophotometer.
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Description
This quiz explores the principles and procedures of protein estimation using the Lowry method. It covers the Biuret reaction and the reduction of the Folin-Ciocalteau reagent, along with the necessary materials and steps for executing the test. Test your knowledge on this essential biochemical technique!
