Protein fractionation

most used methods of protein fractionation

1. 1 WELCOME

2. Protein Fractionation Presented By: Jaspreet kaur Dep. Of Food Science & Nutrition

3. Content : 3 Introduction & Definition of Protein Fractionation Fractionation protocol for Protein Strategy of Fractionation Protein Fractionation Techniques: Conclusion References

4. Introduction Fractionation is a separation process in which a certain quantity of a mixture (solid, liquid, suspension or isotope) is divided up in a number of smaller quantities (fractions) in which the composition varies according to a gradient. Fractions are collected based on differences in a specific property of the individual components. A common trait in fractionations is the need to find an optimum between the amount of fractions collected and the desired purity in each fraction. Fractionation makes it possible to isolate more than two components in a mixture in a single run. This property sets it apart from other separation techniques. 4

5. Introduction Fractionation is a separation process in which a certain quantity of a mixture (solid, liquid, suspension or isotope) is divided up in a number of smaller quantities (fractions) in which the composition varies according to a gradient. Fractions are collected based on differences in a specific property of the individual components. A common trait in fractionations is the need to find an optimum between the amount of fractions collected and the desired purity in each fraction. Fractionation makes it possible to isolate more than two components in a mixture in a single run. This property sets it apart from other separation techniques. 5

6. Protein Fractionation Definition Protein Fractionation is a process or series of processes intended to isolate a single or multiple type of protein from a complex mixture of proteins. 6

7. Fractionation of food Fractionation is also used for culinary purposes, as coconut oil and palm oil are fractionated to produce oils of different viscosities, that may be used for different purposes. Mango oil is an oil fraction obtained during the processing of mango butter. Milk can also be fractionated to recover the milk protein concentrate or the milk basic proteins fraction. 7

8. Fractionation protocol for Protein Prerequisite information about the protein. Protein are diverse in composition structure behaviour, you should know about their origin. As your fractionation strategy depends on it. Where is this enzyme or protein present in the cell?(intracellular, extracellular, membranous). How you can purify this protein in as few steps as possible without the loss of activity.(assayable enzyme activity). Keeping in consideration of temperature and time. 8

9. Strategy of Fractionation Fractionation procedures or steps to isolate protein based on physical characteristics. Characteristic Procedure Charge 1. Ion exchange 2. Electrophoresis 3. Isoelectric focusing Polarity Adsorption chromatography Paper chromatography Reverse phase chromatography Hydrophobic interaction 9

10. cont… Characteristic Procedure Size 1. Dialysis 2. Gel electrophoresis 3. Gel filtration 4. Ultracentrifugation Specificity 1. Affinity chromatography 2. Immunopurification Solubility 1. Salt precipitation 2. Detergent solubilization 10

11. Protein fractionation techniques: Homogenisation Centrifugation Fractionation by precipitation Ultra filtration Chromatographic and Electrophoresis 11

12. Homogenisation Hydrophilic peptides/protein are generally extracted with homogenization in water or in solutions of organic acids whereas organic solvents are used to obtain highly hydrophobic peptides/protein. Homogenization in a mixture of organic solvents (chloroform/ methanol) can be used for peptide extraction as well as for the removal of sample interferences after producing a biphasic system. By using this method, Kostyra et al., (2003 ) studied the opioid activity of cheese and fermented milk samples and some other samples, homogenization in water has been widely applied on cheese, fish, meat, and cereals samples Typically, the ratio of water to cheese used was 2:1 in the homogenization process, followed by an incubation step of an hour at 60°C. 12

13. Conti..... Homogenization Homogenization in a blender and in a Dounce are common and simple procedures form disruption soft tissues such as liver, heart, brain, and muscle or other sample. These methods are rapid (5 to 10 min) and gentle to proteins. These homogenization procedures usually produce heat, and thus the blender and the associated container should be prechilled at 4℃. The homogenization should be performed in a cold room or on ice. 13

14. Cont…. To get a homogeneous solution after getting rid of cell debris, tissues and insoluble stuff either by filtering through muslin cloth or filter paper. This is called Crude extract containing the protein or enzyme of your interest plus a mixture of other proteins. Find out the amount of total protein(By Protein assay) in this Crude extract. 14

15. Protein Assay Lowry method as being the most accurate and sensitive method down to 0.01mg/ mL and widely used . Based on the biuret reaction in which the peptide bonds of the proteins react with copper under alkaline conditions to produce CU +, which reacts with the Follins reagent resulting in strong blue colour which depends partly on aromatic a.acid such as tyrosine and tryptophane . 15

16. Centrifugation The use of centrifugation is one of the simplest methods used for isolation and fractionation of proteins. Centrifugation can be used for different purposes. It can be a first step to separate different cell substructures where our proteins of interest are locally concentrated, for instance, mitochondria, membrane, or nucleus. 16

17. Conti..... This process involves multiple centrifugation steps and, as a result, the cellular homogenate is separated into different layers based on the molecular weight, size, and shape of each component. Afterwards, solubilization steps and enrichment and fractionation steps should be carried out to isolate the protein fraction from the selected layer prior to MS analysis. 17

18. Centrifugation Separate proteins by size or density Differential centrifugation - separates large from small particles Isopycnic (sucrose-density) centrifugation - separates particles of different densities 18

19. Precipitation It is recognized that among the different precipitants the most widespread is ammonium sulphate. The addition of high amounts of this salt or other such as sodium chloride into a protein solution provokes an increase of protein interactions followed by protein aggregation and finally precipitation. This is known as a salting-out process and, as the salt concentration needed for protein precipitation varies from one protein to another, it allows selective protein separation. 19

20. Salt Out Dialysis is commonly used for removing the salt from the proteins. As ammonium sulphate presence in the protein can interfere in many ways. Dialysis -a process that separates molecules according to size through the use of semi permeable membranes containing pores of less than macromolecular dimensions . Pores in the membrane allow solvents, salts and small metabolites to diffuse across but block larger molecules. Cellophane (cellulose acetate) most commonly used dialysis material. Usually used to change the solvent in which the protein is dissolved in. Can also be used to concentrate a protein solution by placement in a polymeric dessicant (PEG) which cannot go through the membrane but absorbs water through the membrane. 20

21. Figure : Use of dialysis to separate small and large molecules. 21

22. Ultra Filtration Ultra filtration is mainly useful for fractionating peptides as well as the removal of proteins and other macromolecules based on their molecular size. Dedicated membranes are mostly made of polysulfone or cellulose derivatives. UF is used extensively in the dairy industry; particularly in the processing of cheese whey to obtain whey protein concentrate (WPC) and lactose-rich permeate.  In a single stage, a UF process is able to concentrate the whey 10-30 times the feed. 22

23. Chromatography 23 Analytical methods used to separate molecules. Involves a mobile and a stationary phase. Mobile phase is what the material to be separated is dissolved in. Stationary phase is a porous solid matrix which the mobile phase surrounds. Separation occurs because of the differing chemistries each molecule has with both the mobile and stationary phase. Chemistries are different depending on the specific method.

24. Chromatography terms The analyte is the substance to be separated during chromatography. The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte . The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components.

25. Gas - Solid : Mobile phase is gaseous, stationary phase is a solid matrix. Liquid - Solid : Mobile phase is liquid, stationary phase is a solid matrix. If separation is based on ionic interaction the method is called Ion Exchange chromatography. If separation is based on solubility differences between the phases the method is called adsorption chromatography. If the separation is base on size of molecule the method is called gel filtration or size exclusion. If the separation is base on ligand affinity the method is called Affinity chromatography. 25 Types of chromatography

26. Some other Important Chromatography 26 Paper Chromatography: In this system a filter paper sheet is used as support for stationary phase. Thin Layer chromatography: In this technique a glass plate, plastic sheet or a piece of metal foil serves as a support for the stationary phase which is applied in the form of a thin layer on these material. Colum chromatography: in this technique stationary phase is packed into a tubular glass or metal column

27. Ion-exchange chromatography Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. It is often used in protein purification, water analysis, and quality control.

28. Cont…. PRINCIPLE Reversible electrostatic attraction of a charged molecule to a solid matrix possessing opposite charge . Elution is done by increasing salt concentration or changing pH . FACTS Proteins possess both (+) and (-) charges At pH=7: Aspartic and glutamic acid have negatively charged side groups Lysine, arginine, histidine have positively charged side groups 28

29. 29

30. Gel Filtration Chromatography Gel filtration (chromatography), is also known as molecular sieve chromatography. Gel filtration chromatography separates molecules according to their size and shape. The stationary phase consists of beads containing pores that span a relatively narrow size range . Smaller molecules spend more time inside the beads than larger molecules and therefore elute later (after a larger volume of mobile phase has passed through the column).

31. Theory Column matrix Porous beads Large molecules are “excluded” from the pores and travel through the column fastest Small molecules are “included” – can diffuse into the pores and elute later

32. Affinity Chromatography Described as the most powerful highly selective method It relies on the ability of most proteins to bind specifically and reversibly to their ligands . Generally used in late purification steps . 32

33. Affinity chromatography 33

34. Column chromatography Column chromatography (CC) is an extremely valuable technique for purification of synthetic or natural products. Compounds are separated by CC through the same mechanism as TLC; through differential intermolecular forces between the components of the mixture with the mobile phase, and between the components with the stationary phase. A variety of adsorbents can be used as the stationary phase; silica gel (which is very polar) is most commonly used in organic chemistry.

35. Polar components (b) adsorb more strongly to the polar silica and elute after the less polar components (a), which move more quickly with the non-polar (relative to silica) solvent. COLUMN CHROMATOGRAPHY

36. Paper Chromatography Paper chromatography - separation of small polar molecules. Mostly used to separate amino acids, oligopeptides . Historically the first chromatography but not really used today. However, principles of its use are useful to know. Rates of migration of the substances are determined by relative solubilities in the polar stationary phase (paper) and the nonpolar mobile phase A given solute is distributed between the mobile and stationary phases according to its partition coefficient K p = concentration in stationary phase concentration in mobile phase Molecules are separated according to their polarities, with nonpolar molecules moving faster than polar molecules 36

37. 37

38. Conti…. After the solvent has migrated an appropriate distance, the chromatogram is removed from the solvent and dried. If not colored, the separated materials can be detected by radioactivity, fluorescence, etc. The migration rate of the substance is expressed by the following ratio: Distance traveled by substance R f = Distance traveled by solvent front Each substance has a characteristic R f value for a given solvent and paper type. 38

39. Thin Layer Chromatography 39 Principle: In thin layer chromatography separation of a mixture is achieved over a thin layer of aluminum oxide or silica gel to which they are adsorbed by different physical forces. Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase.

40. Thin Layer Chromatography

41. Electrophoresis Methods 41 Electrophoresis separates mixtures of proteins based on charge, charge/mass ratio or size. Principle It is the process of moving charged bio molecules in solution by applying an electrical field across the mixture. Biom olecules moved with a speed dependent on their charge, shape, and size and separation occures on the basis of molecular size.

42. When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. Factors influenced electrophoresis mobility: net charge of the molecule size and shape concentration of the molecule in solution

43. Electrophoresis Separation of proteins, nucleic acids, etc. by size, shape, charge Proteins migrate based on their charge-to-mass ratio Proteins visualized (radioactivity or staining) Use gels made of crosslinked polymer ( polyacrylamide ) or solidified agarose Purification of RNA polymerase Steps 1 2 3 4 5 6 43

44. Conclusion 44

45. ANY QUESTIONS? 45

46. References 46 http://www.google.com/search?q=affinity+chromatography+%3B793%3B528 http://www.google.com/webhp?nord=1#nord=1&q=electrophoresis http://www.sigmaaldrich.com/catalog/product/sigma/d9938?lang=en&region=IN Sawhney S.K. and Randhir Singh (2011). Intoductory Practical Biochemistry.9 th Reprint pp 195-216.

47. 47