Isolation and purification of plant viruses

description about how to isolate and purify plant viruses

1. Isolation and Purification
of Plant viruses
N. H. SHANKAR REDDY
Ph.D., Plant Pathology
Annamalai University

2. •The process of separation of virus particles and other chemicals present in the host
constitutes.
•Purification of plant viruses helps in the study of
1.Physiochemical properties of virus
2.Virus morphology (size and shape)

3. Growing and extraction of virus
•Infected leaves are thoroughly homogenized in water or preferably in phosphate, borate or
citrate buffer in an electric grinder or in a mortar with pestle
•Tissue homogenate is strained through a piece of muslin cloth (or cheese cloth).

4. Fractionatingviruscontainingextract
•Crudesapwhichcomesoutandcontainsvirusiscollectedandthenpouredintocentrifuge
tube.
•Thetubeisspunatlow-speed(3000-17000g).Asaresult,thecrudesapdifferentiatesinto
supernatantandapellet.
•Thepelletisdiscardedandthesupernatantwithvirusiscollected

5. •Thesupernatantwithvirusispouredintocentrifugetube.
•Thetubeisplacedinfixed-angle-rotorofultracentrifugeandspunathighspeed(40000-
150000g).
•Afterthetubesettles,thevirussedimentsandformstinypelletatthebottomofthetubeand
asupernatantoverit.
•Supernatantisdiscardedandthepelletofvirusismixedwithabufferandstirredwithrod
sothatitre-suspendsinbuffer

6. Purificationofvirus
•Lowandhighspeedcentrifugationstepsarerepeated2-3timesandthevirusispurifiedby
densitygradientcentrifugation,themostfrequentlyusedtechnique.
•Atieroflayerofsucrosesolutionsofdifferentconcentrations(e.g.,10-40%),andhence
densities,isformedinthecentrifugetube;layeratthebottombeingthemostdenseandone
atthetoptheleastdensewithlayerofintermediateconcentrations.
•Virussuspensionisplacedattopofthetop-mostlayerandcentrifugetubecentrifugedin
swimming-bucketrotorsathighspeedultracentrifuge.

7. •Whensettled,plantvirusparticlesmovetogetherasabandingradientsolutionofsucrose.
•Thevirus-bandiscollectedasseparatefractionthroughpunctureatthebottomofthe
centrifugetube.
•Thevirus-fractionisplacedincellulosedialysistubingandsucroseisremovedbydialysisin
buffersolutionorwater.Thus,thevirusisobtainedinpureform

8. Techniques used in purification of plant
viruses
•Density gradient centrifugation
•Ultracentrifugation
•Salt precipitation or crystallization
•Isoelectric precipitation

9. Video available at you tube
Channel –Geeky Researcher