Owner manual

EBU-222 Instruction Manual, version 7/28/2011
9
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SECTION 4
APPLICATIONS & RUNNING CONDITIONS FOR TANK TYPE ELECTRO-BLOTTING
4.1 RECOMMENDED BUFFERS, MEMBRANES, POWER SETTINGS AND TRANSFER TIMES
Gel Type Blotting Buffer Transfer Membrane Power
denaturing: SDS-PAGE
(Western)
Towbin (25mM Tris/192mM Glycine/ pH 8.3/
Methanol 20% (w/v)
nitrocellulose/nylon PVDF: ion-exchange • room temperature, 70V-100V,
200mA, 1.5- 4 hours
• 4ºC, 150V, 400mA, .5-2 hours
• Overnight, 30V, 75mA
non-denaturing: SDS-PAGE
(acidic or neutral proteins)
25mMTris/192mM Glycine, pH 8.3
25mM sodium phosphate, pH6.5
15mM sodium borate, pH9.2
nitrocellulose; nylon PVDF; ion-
exchange
diazo-paper
diazo-paper
• room temperature, 60V, 200mA, 4
hours
• 4ºC, 125V, 400mA, 1.5 hours
• Overnight, 30V, 100mA
IEF (native-basic proteins,
acid urea gels)
0.7% acetic acid (w/v)* nitrocellulose; nylon
diazo-paper
• room temperature, 70V, 400mA, 4
hours
• 4ºC, 100V, 600mA, 1.5 hours
• Overnight, 30V, 200mA
DNA
(Southern)
1XTAE or agarose
0.5X TBE, acrylamide
nylon; +charged
nylon, neutral or +charged
• room temperature, 30-40V, 25-
150mA, 2-4 hours
• 4ºC, 125V, 400mA, 1.5 hours
RNA
(Northern)
19.6mM phosphate/5.4mM citrate
pH 3.0 or 25mM sodium phosphate pH 6.5
nylon nitrocellulose • room temperature, 60V, 200mA, 4
hours
• 4ºC, 125V, 400mA, 1.5 hours
• Overnight, 30V, 200mA
4.2 REFERENCES
1). Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Seidman,J.G.,Smith,J.A.,Struhl,K.(ed.)(1993).
CurrentProtocolsinMolecularBiology.Vol.2,GreenePublishingAssociates,Inc.andJohnWiley&Sons,
Inc., Ch.10.
2). Burnette,W.N.(1981).Westernblotting:Electrophoretictransferofproteinsfromsodiumdodecylsulfate-
polyacrylamidegelstounmodiednitrocelluloseandradiographicdetectionwithantibodyandradioiodin-
atedproteinA.Anal.Biochem.112:195-203.
3). Peluso,R.W.andRosenberg,G.H.(1987).Quantitativeelectrotransferofproteinsfromsodiumdodecyl
sulfatepolyacrylamidegelsontopositivelychargednylonmembranes.Anal.Biochem.162:389-398.
4). Perides,G.Plagens,U.,andTraub,P.(1986).Proteintransferfromxed,stainedanddriedpolyacryl-
amidegelsandimmunoblotwithproteinA-gold.Anal.Biochem.152:94-99.
5). Tesfaigzi,J.,Smith-Harrison,W.,andCarlson,D.M.(1994).Asimplemethodforreusingwesternblotson
PVDFmembranes.Biotechniques.17:268-269.
6). Towbin,H.,Staehelin,T.,andGordon,J.(1979).Electrophoretictransferofproteinsfrompolyacrylamide
gelstonitrocellulosesheets:Procedureandsomeapplications.Proc.Natl.Acad.Sci.U.S.A.76:4350-
4354.
7). Sambrook,J.,Fritsch,E.F.,Maniatis,T.(1989).MolecularCloning.ALaboratoryManual.2nded.Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 18.47-18.61.