Protein_analysis using ppt Teknik mantap.pptx

Protein analysis

1. Protein analysis 02/09/2014 by TDLV 1 Progress in science depends on new techniques, new discoveries and new ideas , probably in that order. Sydney Brenner Tools for protein analysis

2. 02/09/2014 by TDLV 2 Why do we analyse proteins? Explore how amino acid sequences specify the structure of proteins How proteins bind to substrates and other molecules to perform their functions. Understand the function of the protein based on its structure.

3. Determing Primary Structure of an Entire Protein

4. 02/09/2014 by TDLV 4 Main Steps in Protein Analysis Extraction of proteins. Purification of the protein. Structural Characterization of the protein.

5. 02/09/2014 by TDLV 5 Protein Characterization Characterization of proteins and peptides : 3 different processes: Determining the Amino Acid Composition finding out the amino acids that make up the protein and their number. Determining the Amino Acid Sequence finding out the sequence of amino acids of the proteins in their order. Determining the Molecular mass of the Protein

6. 02/09/2014 by TDLV 6 Determination of Amino Acid Composition peptide  hydrolyzed  a mino acids by heating in 6M HCl at 110ºC for 24 hrs . The amino acids  separated by HPLC. measured by reacting them with ninhydrin . Alpha Amino acids  intense blue color , imino acids ( proline , hydroproline )  yellow color. The concentration of a single amino acid is proportional to the light absorbance of the solution after adding ninhydrin . The elution profile of the amino acids is obtained. The identity of the amino acid is revealed by its elution volume

7. 02/09/2014 by TDLV 7

8. 02/09/2014 by TDLV 8 Determination of protein Molecular Mass by Mass Spectrometry Mass Spectrometry: include an ionization source, a mass analyzer and a detector. The ion hits the mass analyzer. The time the ions take in flight is recorded by triggering a laser pulse. It depends on one principle The smaller ions will fly faster and hit the detector first then the larger ions reach. The ions hit the detector in order by mass.

9. 02/09/2014 by TDLV 9 Chromatography for protein purification

10. 02/09/2014 by TDLV 10 Chromatography-3 matrices

11. 02/09/2014 by TDLV 11 Protein purification by chromatography

12. 02/09/2014 by TDLV 12 Genetically-engineered tags provide an easy way to purify proteins - antigenic determinant, or epitope, recognized by a specific antibody - short string of histidines (His-tag) binds to an affinity column containing immobilized nickel ions - Glutathione S- transferase ( GST) fusion protein binds to an affinity column containing glutathione

13. 02/09/2014 by TDLV 13 Purification of protein complexes by using a GST-tagged fusion protein

14. 02/09/2014 by TDLV 14 Protein analysis SDS PAGE (   sodium dodecyl sulfate polyacrylamide-gel electrophoresis Protein separation

15. 02/09/2014 by TDLV 15 SDS PAGE

16. 02/09/2014 by TDLV 16 Separation of proteins by isoelectric point

17. 02/09/2014 by TDLV 17 Specific proteins can be detected by Western blotting

18. 02/09/2014 by TDLV 18 The diffraction of X-ray by protein crystals can reveal a protein’s exact structure