...cOnTiNue...
4th lecture...precaution when handling protein...proteolytic degradation - protein broken apart by proteases that come from lysosome where by when cell disrupted lysosome breaks n release protease which can damage the other protein thus minimized the activation of protease to avoid protealysis, oxidation - sulfur group from cysteisine undergo oxidation forming disulphide bond that are not normally presence n by reducing agent like dithiothreitol or beta mercaptoethanol was added to prevent undesirable disulphide bond, dilution - protein stick to surface by reducing available activity so to prevent significant loss not store dilution solution for too long..fractional salt precipitation - method for concentrating the protein as well as crude separation from other protein..every protein has their own solubility limits bcoz solubility affected by concentration of da protein of interest, pH n temperature... column chromatography is a revolutionary event in protein separation for obtaining da pure protein...different kind of chromatography depends on size of protein, net charge, specific ligand binding properties n hydrophobicity...column chromatography has 2 type of components which are solid phase n liquid phase..basic step is that da sample is loading, washing and then elution...
gel filtration chromatography - a.k.a molecular sieve chromatography / size exclusion chromatography...stationary/solid phase separate protein based on size n shape..volume of bufer required to elute a specific protein depends mostly on molecular weight of the protein n shape play important role so elution process need to follow hydrodynamic volume...
ion exchange chromatography- charged group covalently attached to stationary phaseeither +ve / -ve...protein bind to da matrix by electrostatic interactions..this interaction depends on net charge on da protein n salt concentration of da buffer...higher net charge more sticky to da opposite matrix n higher salt concentration required to elute it from column...
cation exchange - matrix has -vely charge groups
anion exchange - matrix has +vely charge groups
liquid chromatography - hydrophobic interaction chromatography (HIC) n reverse phase chromatography (RPC)...separation that exploit the hydophobic or non- polarity properties of protein n molecule...high performance liquid chromatography - high pressure pumpng overcome the resistance to flow through column..it requires non-compressible resins n strong column which then resulting quicker than conventional gravity flow column, achieve better separation of closely similar compound, the resolving power increase with column length n powerful n versatile form of chromatography ..
5th lecture...protein electrophoresis...in electric field protein or other charge molecule will move with velocity depends on direction of the charge n inversely on its size n shape..pH is important to determined the net charge..
gel electrophoresis - carried out supporting media with pores that big enough to allow passage of macromolecule..electric charge is applied to molecule moves forward electrode opposite to their charge but slow down by gel..larger shape move slowly while small molecule moves faster..proteins in gel easy stained for detection due to the net charge n molecular weight..
discontinuous gel electrophoresis - 2 gel layer ; lower resolving gel, upper stacking gel ; buffer used to separate 2 gel layers of different ionic strength n pH.; staking gel has lower acrylamine concentration so pore size is larger..
sodium dodecyl sulfate - poly acryamide gel electrophoresis (SDS-PAGE) - a variant of electrophoresis in which the buffer contains SDS, a detergent bind to the protein... sample need treatment which are B-mercaptoethanol (no sulphide bond) n heating to ensure complete unfolding n complete separation of different polypeptide..large -ve charge resulting bound of SDS masks the native charge on the protein so all protein have essentially da same charge to mass ratio n same shape..rate of movement depends only m.w of individual polypeptide chain...protein mobility inversely proportional to da log of the mass of individual polypeptide chain...function to estimate purity, determined molecular weight of individual polypeptide subunits n purification of small amount of polypeptide for sequence analysis
isoelectric focusing - separation based on isoelectric point (PI) on charge difference..ph gradient set up first n the mixture of molecule is applied electric field is turn on n each protein moves to position of net charge at zero...
2D electrophoresis - isoelectric in 1st dimension n followed by SDS-PAGE at 90 degree to 2D..
protein primary structure determination - amino acid sequence, nucleotide sequence, amino acid residues n sequence comparison of protein...important of amino acid side chain; hemoglobin is the oxygen transport protein in blood which presence in oxy / deoxy, mutant hemoglobin are known to exist, sicle cell hemoglobin...amino acid composition- fundamental characteristics of any protein which total acid hydrolysis that consist of aqueous acid n amino acid in hydrolysate quantitated using ion exchange...
amino acid sequence determination - amino terminal residue n edman degradation...amino terminal residue (NH2) can be determined by derivatization of whole peptide or protein with either 1-fluoro-2,4-dinitrobenzene FDNB or reagent that gve fluorescence derivatives..total acid - derivation N-terminal residue , y-amino derivatives...Edman degradation - only one residue at a time from amino N-terminal can be chemically derivative, removed n identified...repeated step derivation...steps; coupling, cleavage, conversion n sequencing...problem; when proteolysis has been accomplished the peptide separated n sequenced - solution ; sequence of 2 different cleavage methods r examined for overlaps...
6th lecture...affinity chromatography...based on specific ligand binding to protein of interest ...affinity ligand covalently attach column material, ligand can include; substrate analogs, inhibitors, natural n artificial ligand, cofactor, metals, binding proteins n anythings that utilize biologically for genetically engineered protein n fusion protein...column preparation include; ligand attach to matrix, linker or spacer arms, matrix uncontaminated, covalent attachment n binding should be specific...elution - buffer containing free ligand or destabilizing binding or make it weak by change pH/ionic strength n chaotropic/ denaturing agents...common example of affinity systems ; immunoaffinity chromatography - antibody specific for protein used to purified specific protein n immobilised metal affinity column (IMAC) - fusion protein with polyHis tags, can be purified in column n imidazole group on the end of the recombinant protein bind with high affinity...
No comments:
Post a Comment