Molecular Staining

Molecular SinvingMedia such as polyaoylamide ,agar stauch haveCross linked Structura giving rise to pores within thegel beads.polyacrylamide / agaiose I starch The larger moleculesalso all made to squuge through the pores Thesmaller molecules pass through the pares earityLargernare retailsdBufferThe buffer can affect the electrophoretic mobilityof the sample in various ways.Composition:- Commonly used buffer are formateacetate , citrate, phosphate of EDTA , autate pyricline,Tris and Barbitone etc.The choice of buffer depends upon the type ofSample being electrophoresedlonic Strength:-Measure of the electrical environment of ions in aSdn.when a increase Lonic strength of the buffer means a laugershare of the current being cavied by the buffer ions8 meager (small quality) proportion caused by thesample ions.whenionic Strength , a larger Share of the currentbeing cauld by the sample ions leading to a fasterseparation.1-1 Note: lonic strength is usually b/w 0.05 - O.IM PHdetermines the degree of ionization of organiccompounds and it can affect the rate of migration.when PHt ionization of organic awds.PHV11,of 11 basesEg : an ampholyte (aa) - The aa has both acidic &basic propertiesComponents :-a power source with volt meter and voltage regulatorelectropharesis tark that holds electrophoresis bufferan anode and a cathode connected with power source.a glass plate holds the gela combvoltage suppliedCurrent follow flowing blu the cathode cy ancle establishan electric field in the electrophorius tank and in thegel which will be used for Separating compounds accordingto their change or sizeGelcolloid in solid form (94% H20)Separationbased on moleculcu sievingpurous yel acts as Sieve by retarding or in some casesby obstructing the movement of mauomoleculee while allowingsmaller molecules to migrate freelyagawose, polyawylamide - widely used.that gel media is electrically neutral.lgar gelNAPolyavylamide - pro, pep, a a . samples travel through a running buffer when ane field is applied.Separation is stopped before analytis come to the endof the supportDistance traveled is called migration time (dm) whichcan be compared to standardsSample is placed on the gel support then in e field.Multiple amples are applied and allowed to separateas they migrate toward the ends of the gelThe location of the migration band ( intersity )isdetumeredThe Support may be horizontal 1 vertical when arunning buffer that causis CurrentTo avoid broadf bandRunning gelSupport used for separation in samplestacking gelClarge pure size) . This is where wells are located.has a low degree of cross listingwhen e field applied the analytes travel quickly to theboundary of the running gel. Then they slowly traveland other pauts begin to catch up, resulting innarrowerbands and formation of more concertracted bands.Detection :-Direct examination of analyte on gel afle treatmentwith dye / stair ( for pro amide black , CBB)concertrations.Silver N2 is used in Silver staining f or low