Estimation of DNA by Diphenylamine method

Hello guys ^_^

Long time since a Chemical estimation done and that is why we will be discussing DNA estimation today. This can be done by two methods, one of which we will be learning today. That is Diphenylamine method (DPA method).

Let's do it.

ESTIMATION OF SUGAR USING BENEDICT’S METHOD

ESTIMATION OF REDUCING SUGAR USING BENEDICT’S METHOD 

AIM: 
 To estimate the amount of glucose present in the given unknown solution using Benedict’s quantitative reagent. 

PRINCIPLE:
It uses Reagent called  Benedict’s quantitative  reagent which contains copper sulphate-sodium acetate and sodium carbonate. It also contains potassium thiocyanate and small amount of potassium ferricynate (prevents the pre oxidation of copper). Methylene blue will be used as an additional indicator. The reduction of Cu3+ ions by sugar is a nons tiochemetric equation and is only constant over a small range of sugar concentration. To obtain accurate results the volume of sugar added must be within 6-12ml for 10ml of Benedict’s regent.On the reduction of Cu3+ ions, which inhibits the end point of the titration digest the transition from blue to white to be readily observed.

REGENT REQUIRED:

•  2mg/ml glucose
• Benedict’s quantitative reagent (sodium citrate, potassium thiocynate ,18%  sulphate and 5% potassium ferricyanide )
• Anhydrous sodium carbonate

PROCEDURE: 

5ml of Benedict’s reagent was pippeted out into a clean conical flask. Anhydrous sodium carbonate was added to provide the required alkalinity with a few porcelain bits and heated to boiling over a moderate flame. Standard glucose solution is taken in a burette. When the Benedict’s solution boils continuously, glucose solution is added drop by drop (1 drop/sec) till last trace of blue colour disappears. The volume of glucose rundown is noted and the titrations are repeated to concordant values.  Then the burette was filled with unknown sugar solution and the Benedict’s reagent was titrated as before. The volume of sugar solution rundown was noted and titrations are repeated for concordant values. 

RESULT:

A light Green, Yellow or Brick color

-Dixy

Protein estimation by Folin Lowry's method

Hello everyone,

Today I will be talking about  the most important type of chemical estimation for Proteins ie Folin lowry. The Lowry assay  is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and so on. The method is sensitive down to about 10 µg/ml and is probably the most widely used protein assay despite its being only a relative method , subject to interference from Tris buffer, EDTA, nonionic and cationic detergents, carbohydrate, lipids and some salts. The incubation time is very critical for a reproducible assay. The reaction is also dependent on pH and a working range of pH 9 to 10.5 is essential.

The determination of protein concentration is an essential technique in all aspects of protein studies and proteomics. 

Requirements:

• 2% Na2C03 N NaOH
• 0.5% CuSO4 solution in 0.1% Na- K tartarate solution(Alkaline CuSO4)
• Folin -ciocaltaeau reagent
• Std protein (BSA)
• Distilled waater
• Unknown protein solution

The Folin–Ciocalteu reagent (FCR) or Folin's phenol reagent or Folin–Denis reagent, also called the Gallic Acid Equivalence method (GAE), is a mixture of phosphomolybdate and phosphotungstate . It is named after Otto Folin, Vintilă Ciocâlteu, and Willey Glover Denis.

The basic principle behind the experiment is very simple

The Nitrogen group present in protein reacts with copper ions under alkaline conditions and subsequent reduction of FC reagent to blue colour compound (heteropolymolybdenum blue)

Assay

1. Prepare a series  dilutions of BSA solutions are prepared by mixing stock BSA solution and water in the test tube  
2. Add 5.5 ml of Alkaline CuSO4 solution reagent in each tube.Mix the solutions well.
3. Incubate the tubes  for 10 min at RT, then cool to room temperature.
4. Then add 0.5 ml of reagent Folin Ciocalteau solution  to each tube and incubate for 30 min. 
5. Zero the colorimeter with blank and take the optical density (measure the absorbance) at 660 nm.
6. Measure absorbance at 650 nm .
7. Plot the absorbance against protein concentration to get a standard calibration curve.
8. Check the absorbance of unknown sample and determine the concentration of the unknown sample using the standard curve plot.

-Dixy

Protein estimation by Biuret Test

Heya lovelies

So chemical estimations have been my favorite in practicals. Hence today we will be discussing estimation of proteins by Biuret test. Biuret test can be used as a qualitative or quantitative method for protein estimation.

Lets first understand what is qualitative and quantitative.

Qualitative - whether the particular compound is present or no.

Quantitative - if present, then how much.

Biuret reagent is prepared with sodium hydroxide and hydrated copper sulphate with potassium sodium tartarate.

Mnemonic: Katy Perry danced to a Pole Song.

Standard used - Bovine Serum Albumin (BSA).

Proteins tend to bind Cu ions in alkaline conditions to form copper chelate in basic pH, which is blue in color. This color is measured at 540 nm in a colorimeter. Potassium sodium tartarate is known to stabilize Cu ions.

Biuret estimation though is very accurate, is not sensitive enough. it cannot measure proteins concentration below 100 mcg.

Folin Lowry is an alternative method used for lower protein containing samples.

That's all.

Hope it helps you learn.

-Staph <3