Qualitative and quantitative techniques of protein analysis

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 biology.

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.

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 1-25 µg protein.

8. 2) Bicinchonic acid method.

9. 3) Lowry’s method

10. 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.

11. 5) Absorbance at 280nm.

12. 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.

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

14. 2) NATIVE GEL ELECTROPHORESIS.