B-I&feature=related.

Slides:

Advertisements

Similar presentations

Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry

Advertisements

Membranes membranes are composed of a lipid bilayer and associated proteins ~1/3 of a cell's proteins are directly associated with membranes many soluble.

Ameer Effat M. Elfarash Dept. of Genetics Fac. of Agriculture, Assiut Univ. Gene Expression.

PURIFICATION OF GFP USING HIC CHROMATOGRAPHY. Chromatography  A technique used to separate molecules based on how they tend to cling to or dissolve in.

Umair Saleem Methods in protein chemistry Hydrophobic interaction chromatography.

Protein Purification Molecular weight Charge Solubility Affinity.

Chromatography for Protein purification 1

ION EXCHANGE CHROMATOGRAPHY PREPARED BY- MD.MARUF HASSAN.

LABORATORY 6: PURIFYING THE FLUORESCENT PROTEIN 2014.

Protein Purification What is protein purification?

Protein Purification Strategies Course: Methods in protein chemistry Rahman M. Mahfuzur 2012/01/11 SLU.

Review: Amino Acid Side Chains Aliphatic- Ala, Val, Leu, Ile, Gly Polar- Ser, Thr, Cys, Met, [Tyr, Trp] Acidic (and conjugate amide)- Asp, Asn, Glu, Gln.

Protein Purification and Analysis Day 4. Amino Acids, Peptides, and Proteins.

Metal Chelate Affinity Chromatography Wenbo Dong Yagmur Yagdiran.

Protein Purification and Analysis Solubility of proteins important for purification: 60-80% soluble, 20-40% membrane Size of proteins varies Some proteins.

Protein Purification. Why purify Proteins? Characterize Function Activity Structure Study protein regulation and protein interactions Use in assays Produce.

PRESENTED BY:- GAIKWAD OVESH & MD.NADEEM MANSOORI

18.7 Isolation, Purification, and Fractionation of Proteins (1)

Chapter 4-1 Chromatography

Chapter Five Protein Purification and Characterization Techniques

Introduction to Proteins as Products Biotechnology 2.

Types of Mechanism in the Chromatography

Biochemistry February Lecture Analytical & Preparative Protein Chemistry II.

Table 5-1 Protein Purification Essential for characterizing individual proteins (determining their enzymatic activities, 3D structures, etc.) Two main.

Protein Purification and Characterization Techniques

Analysis of Proteins and Peptides Amino acid composition Molecular weight Isoelectric point Subunit structure Prosthetic groups Solubility Biological activity.

Green Fluorescent Protein (GFP) Purificaiton. Recombinant Cell.

BIOCHEMICAL METHODS USED IN PROTEN PURIFICATION AND CHARACTERIZATION

BIOMAN K. Lampe, MC3 U P S T R E A M D O W N S T R E A M B I O P R O C E S S I N G biosciences.com.

AMINO ACID ANALYSIS. Amino Acid Analysis  Amino acid analysis is the determination of what types of amino acids and how many of each compose a protein.

Techniques in Protein Biochemistry Stryer Short Course Chapter 5.

Lab 6 Salt fractionation In aqueous environments salt ions interact strongly with their complementary charge on a water molecule If the concentration of.

Salting in and Salting out of proteins and Dialysis

Proteomics The science of proteomics Applications of proteomics Proteomic methods a. protein purification b. protein sequencing c. mass spectrometry.

CHROMATOGRAPHY (DEMONSTRATION) Mrs. Chaitali Maitra

PROTEIN TECHNOLOGY By DR ZARINA ZAKARIA. Why to exploit protein Information about protein structure has led to a deeper understanding of the evolutionary.

Intended Learning Objectives You should be able to… 1. Give 3 examples of proteins that are important to humans and are currently produced by transgenic.

Separation techniques ?. Molecules can be separated: Chemically: by charge, by action with specific reagents Physically: by solubility, by molecular weight,

Saida Almashharawi Basic Biochemistry CLS 233 Ch4: Protein Purification 1.

Quiz Next Tuesday (09/18). Figure 4.18 Cation (a) and anion (b) exchange resins commonly used for biochemical separations.

Introduction to Instrumental Analysis - Chromatography

Protein Purification for Crystallization Dr Muhammad Imran Forman Christian College (A Chartered University) Dr Muhammad Imran Forman Christian College.

HISTORY OF CHROMATOGRAPHY AND VARIOUS TYPES OF LIQUID CHROMATOGRAPHY CHROMATOGRAPHY IS A SET OF LABORATORY TECHNIQUES INTENDED TO SEPARATE COMPOUNDS FROM.

Food Analysis Lecture 18 (03/27/2012) Basic Principles of Chromatography (3) Qingrong Huang Department of Food Science Read Material: Chapter 27, page.

Purification of immunoglubin by ion exchange chromatography Bahiya Osrah

Lab 6 Ig Purification What will the Ig be used for? How pure does it need to be? What is the source of the antibody (serum, ascites, cell culture supernatant)

Chromatography PlanarColumn Paper TLC (Thin layer chromatography)

Principles of chromatography

Tymoczko • Berg • Stryer © 2015 W. H. Freeman and Company

 Laboratory technique for the Separation of mixtures  Chroma -"color" and graphein - "to write”.  Colour bands - separation of individual compounds.

Bioseparation II Chromatography Techniques. Chromatography Most widely used purification technique used for biomolecules. Most widely used purification.

Chromatography 1 Lecture 10 An introduction. What is CHROMATOGRAPHY ? Chromato g raphy.

Free Powerpoint Templates Page 1 Free Powerpoint Templates Chromatography by Creative Biomart.

Lab Activity 11 Purification of LDH Part II

Lecture 14 February 23, 2016 Biotech 3.

Lab Activity 12 Purification of LDH Part II

Purification Of Proteins.

BSB Biomanufacturing CHAPTER 13 GMP – Downstream Processes

Purification of Green Fluorescent Protein

Chapter 5. Protein Purification and Characterization Techniques

Column Chromatography

Affinity Chromatography

Ion exchange chromatography By Mennatallah Abdelshaheed.

Lab Activity 11 Purification of LDH Part II

M.S COLLEGE OF ARTS, SCIENCE, COMMERCE AND B.M.S

Purification of Green Fluorescent Protein

Experiment 3 Separation of proteins by Anion exchange chromatography

Chromatographic Techniques

Presentation transcript:

B-I&feature=related

Metabolism0.1 mM, 1x 10 8 Ribosomes 10  1x 10 7 Kinases Cyclins 1  1x  1x 10 5 Transcription factors 10 nM, 1x 10 4 Synaptic Markers 0.1 nM, 1x 10 3 Cytoskelatal Proteins mM, 1x 10 9 copies/cell Are We Ready for Mammalian Proteomics ?

Protein Purification Grow cells in media (vector+tag) Centrifuge, Collect the pellet Lyse the cells (appropriate buffer) Purification Strategy SDS PAGE, Assay Solubility Aggregation Recombination Characterization Mass Spectroscopy X-ray Crystallography Functional Assay

OUTLINE Chromatography techniques Affinity Chromatography (AC) Hydrophobic Interaction Chromatography (HIC) Ion Exchange Chromatography (IEC) Gel Filtration (GF) Capillary electrochromatography (CEC) Other New Strategies for Protein Purification Solubility, Aggregation and Re-folding of Proteins Invented by a Russian botanist named Mikhail Tswett in He separated plant pigments using glass columns packed with calcium carbonate.

OUTLINE Chromatography techniques Affinity Chromatography (AC) Hydrophobic Interaction Chromatography (HIC) Ion Exchange Chromatography (IEC) Gel Filtration (GF) Capillary electrochromatography (CEC) Other New Strategies for Protein Purification Solubility, Aggregation and Re-folding of Proteins Invented by a Russian botanist named Mikhail Tswett in He separated plant pigments using glass columns packed with calcium carbonate.

Affinity Chromatography Surface bound with Epoxy, aldehyde or aryl ester groups Metal Interaction Chromatography Surface bound with Iminodiacetic acid + Ni 2+ /Zn 2+ /Co 2+ Affinity Chromatography (Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

Metal Interaction Chromatography (AC) Points to Note: 1.Avoid chelating agents 2.Increasing incubation time 3.Slow gradient elution (

Biopolymer (phenyl agarose - Binding Surface) Driving force for hydrophobic adsorption Water molecules surround the analyte and the binding surface. When a hydrophobic region of a biopolymer binds to the surface of a mildly hydrophobic stationary phase, hydrophilic water molecules are effectively released from the surrounding hydrophobic areas causing a thermodynamically favorable change in entropy. Temperature plays a strong role Ammonium sulfate, by virtue of its good salting-out properties and high solubility in water is used as an eluting buffer Hydrophobic Interaction Chromatography Hydrophobic region (Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

Fractogel matrix is a methacrylate resin upon which polyelectrolyte Chains (or tentacles) have been grafted. (Novagen) Ion Exchange Chromatography Globular Protein Deformation due to interaction with conventional ion exchanger Maintenance of conformation while interacting with tentacle ion exchanger (

Gel Filtration (

Immuno Affinity Chromatography (

Edman Sequencing

Carboxypeptidase

Carboxypeptidase Mechanism