Explains about the qualitative and quantitative techniques for protein estimation.

1. QUALITATIVE AND
QUANTITATIVE TECHNIQUES OF
PROTEIN ANALYSIS.
SUBMITTED TO : Dr. R.
Rukkumani.
Submitted by : Karishma
Shaw.
Department of biochemistry and molecular biolog

2. CONTENTS
 INTRODUCTION
 STANDARD PATTERN FOR PROTEIN ASSAYS.
 QUANTITATIVE METHODS OF PROTEIN ANALYSIS
 Dye binding method
 Bicinchonic assay
 Lowry’s method
 Biuret method
 Absorption at 280 nm.
• QUALITATIVE METHODS OF PROTEIN ASSAY.
 One dimensional electrophoresis
 Native gel electrophoresis.
 Isoelectric focussing.
 Two dimensional electrophoresis.
 Immunoelectrophoresis.

3. INTRODUCTION
 The determination of amount of protein present in
a solution is a widely used procedure in
biochemistry.
 Important to know the amount of protein in
different stages of purification to calculate the
specific activity of the protein.
 When dealing with a complex protein mixture
such as cell lysates in a purification scheme the
methods used are generally comparative.
 Some methods are dye binding, bca, biuret,
bradford etc.

4.  The most commonly used reference protein is
BSA.
 The standard pattern of protein assays is shown :

5. The standard pattern for protein
assays.
• Preparation of stock solution of reference protein.
• Construction of calibration curve for reference
protein.
• Determination of unknown protein using calibration
curve.

6. QUANTITATIVE METHODS OF
PROTEIN ASSAY.
 1) DYE BINDING METHOD:
 Absorbance spectrum of coomassie blue dye
undergoes a change when it binds to proteins.
 The protonated form of the dye has an orange /
brown appearance and on binding a protein , it
gets deprotonated and its colour changes to deep
blue – detected at 595nm.
 The unprotonated form results from the dye-anion
inyeracting with side chain NH3 groups.

7.  Nh3 group content varies from protein to protein
and hence the colour varies too.
 It is a sensitive method which is suitable over a
range of 1-25µg protein.

8. 2)Bicinchonic acid method.

9. 3) Lowry’s method

11. 4) Biuret method
 The peptide bonds of proteins react with Cu 2+
ions present in biuret reagent under alkaline
conditions to produce a purple colour that can be
detected by measuring at A 540.
 Reaction is sensitive over a range of 0.5-5mg.
 While most standard reagants do not interfere
with this asaay, ammonium ions must be avoided.

13. 5) Absorbance at 280nm.

14.  Protein absorb radiation in the near UV due to
their tyrosine,tryptophan, and to a lesser extent
cysteine,content.
 Hence conc of proteins are estimated by
measuring the absorbance at 280nm.
 An accurate A280 measurement should be
between 0.1 – 1.

15. QUALITATIVE METHODS OF
PROTEIN ESTIMATION
 1) ONE DIMENSIONAL SDS PAGE.

17. 2) NATIVE GEL
ELECTROPHORESIS.