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By: Preet Sidhu (1299064) Teacher : Greg D’Cruz
Boey, C.G. et.al1976, ‘Purification of Tapioca Leaf Rhodanese’, Phytochemistry ,vol.15,pp.1343-1344. (Lecture) Fountoulakis, Dimitraki 2008,’ Protein fractionation by Preparative Electrophoresis’Methods Mol Biol.vol.424,pp.301313. (Available on PUB MED-Student web) Jérôme Zoidakis,2003, ‘Application of preparative electrophorsis for clinical proteomics in urine: Is it feasible’ vol.28,pp.268273 accessed on 5 March 2013 from http://www.dmbj.org.rs/jmb/pdf/2009-4/7.pdf
Electrophoresis is the method for analysis and separation of macromolecules such as RNA. .DNA and proteins and their fragments based on the charge and size-Biological material processed.
The method is particularly efficient for the enrichment of lowmolecular-mass gene products. Proteins are separated according to their size in a cylindrical gel in the presence of an ionic detergent.Preparative Electrophoresis Preparative electrophoresis is a protein fractionation approach useful for the enrichment of low-abundance gene products. .
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The separation membrane is selected so that the proteins of interest can transfer through the membrane away from the larger molecular weight contaminants. .Modern Principles: Size: Separation by size is dependent on the molecular weight of the proteins and the pore size of the membrane contained within the separation cartridge. or alternatively the contaminants are able to move through the membrane leaving the larger protein of interest in the first stream.
. and if the pH is below the pI. A negative charge is placed on a protein at a pH above the protein’s pI. Once power is applied a subset of proteins will rapidly transfer through a large pore size separation membrane towards the opposite electrode. the protein will be positively charged. By manipulation of the buffer pH.Modern Principles Separation by charge is based on the pI of the proteins. an appropriate charge is induced on the protein's of interest.
: rod/slab column Proteins Biological Materials: Polyclonal and monoclonal antibodies SDS-page Polyacrylamide gel electrophoresis Isoelectric focusing Commonly used: .g.System Horizontal/flatbed Vertical system e.
Columns Horizontal Bed Protein load about 100-50ng Protein load of about greater than 50 mg is added Suitable in both types of protein Support is horizontally cast Suitable for faster migration of proteins Support is cast in column due to eluting mechanism Used in : •PAGE(Polyacrylamide gel electrophoresis) •SDS (sodium dodecyl sulfate polyacrylamide gel electrophoresis) Used in: •IEF(Isoelectric focusing) .
CONDITONS: Buffer concentration Voltage .Specific aim & Conditions AIM: Fractionate and purify nanogram to gram quantities of proteins or nucleic acids by electrophoresis.
Nobel Prize 1948).Denatured-PAGE or SDS-PAGE Separation is based upon the molecular weight of proteins. size.PAGE can be classified according the separation conditions into: 1. and shape of macromolecules.Native-PAGE: Separation is based upon charge. The most common method for determining MW of proteins Very useful for checking purity of protein samples . Useful for separation and/or purification of mixture of proteins This was the original mode of electrophoresis (introduced in 1930s. 2.
became popular as a part of proteomic techniques The charge of molecule changes with the pH of its surroundings. Recently. A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the negative electrode. Isoelectric focusing is a technique for separating different macromolecules by the difference in electric charge. Isoelectric focusing is the electrophoretic protein separation on the basis of the mobility at a particular pH The proteins will migrate to that pH which coincides with the pI value .
Samples are electrophoresed (native or SDS PAGE) through a cylindrical gel Bands pass through a thin frit within the elution chamber Isolated bands are drawn radically by a pump onto a fraction Collector into discreet liquid fractions .
the enrichment of low abundance proteins and the detection of low MW proteins and peptides .Benefits Mostly used urine and blood samples for testing (Based on scientific papers) Serve as an ideal means for the construction of proteomic maps.
Companies in Australia Gradiphore- The preparative electrophoresis system: GRADIPHORE Bio-Rad-Preparative page cells and preparative ISF cells .
from http://www. http://staff.co. Phytochemistry .mastbio. Fountoulakis. ‘Purification of Tapioca Leaf Rhodanese’.p df.al1976.The preparative electrophoresis system gradiflow.pdf . Gradiphore. Electrophoresis.nus.rs/jmb/pdf/2009-4/7.edu n.sg/~dbsyhh/Lect%207%20electrophoresis. Accessed 26February 2013.301-313.pp.268-273 accessed on 5 March 2013 from http://www.2003. Boey.org. ‘Application of preparative electrophorsis for clinical proteomics in urine: Is it feasible’ vol.kr/root/product/life/ps/gradiflow/pdf/MB-04PreparativeElectrophoresisSystem.science.vol.nus.dmbj.G.science. C. Dimitraki 2008.28. staff. accesed on 5 march 2013.ND.d.pp.pp.pdf Jérôme Zoidakis.15.424. et.1343-1344.vol.’ Protein fractionation by Preparative Electrophoresis’Methods Mol Biol.edu.