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"O *A*\., with (Fig- Coomassiebrilliant Blue 1 \Ar*.H'cre Hols H Ure 1)

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A Convenient Procedure For storage, PAGE gels are often ide (PVDF) membranesare commonly treatedin one of two ways. They can be usedmatrices for transferblotting (12). for Transfer Blotting of placedin sealedplastic bags containing Here we describe a convenient, in- CoomassieBIue Stained 7Voaeueous acetic acid (9). More com- expensive method for long-terrn non- monly, these gels are photographed photographic storage of the informa- Proteins from PAGE and dried. In lieu of storage,when fur- tion present in electrophoresisgels Gels to Transparencies ther manipulationof the proteinsin the stainedwith dyes basedon blotting the gels is to be done,the patternis trans- stainedgels onto commercial transpar- encies of the type used in preparing ABSTRACT ferred to blotting membranes. Transfer blotting of proteins from viewgraphs for overhead projection. Proteins stained with CoomassieBril- PAGE gels to other films hasbeen ex- The dye-stained transparenciesfaith- liant Blue 1 were transferred effectively by aminedpreviously (7). Electroblotting, fully replicate the information present blotting frctm polvctcryktmidegel electro- diffusion and convection blotting are in the originally stained gel. They are (PAGE) phoresis gels to transparenciesof three common techniquesfor transfer convenientto store.They are also ex- plain-paper the Npe used in copiers. The of proteins (7). Nitrocellulose mem- cellent substratesfor use with gel scan- detuils rl' the original electropherogram branes, diazotrzedpapers, nylon-based ners, since they are flat, dimensionally were retained on transfer and did not fade membranesand polyvinylidene fluor- stable,and haveno color themselves. over a period of three years. Both the pro- tein and the associateddye transfer; how- ever, protein does not transfer in the ab- .\ence of dye. Prolein patterns present in several Qpes of gels-sodium dodecyl sul- .-*(]-so'tr' fute (SDS)-PAGE, non-denaturing PAGE, isoelectric focusing PAGE and SDS- ogarose-al I t ransferred succe s sfully after staining w'ith dve I. Proteins visualized w'ith other organic dltes such as Fast Green FCF 2. Uniblue A 3 and Procion HN- \ \fir Blue MX-R I also transferred,but proteins stained with Stains-all 5 or silver did not. ttl This transfer provides a simple, economi- Y \>*' cal way to preserve data from slab gel ov/ electrophoresisand a convenient methnd to display data using an overhead projec- CoomassieBrilliant Blue R250,l FastGreen FCF,2 tor. The blotted transparencies are also excellent substratesfor use with gel scan- ning densitometers. INTRODUCTION Polyacrylamidegel electrophoresis SOyNr. so3H (PAGE) is widely usedfor analysisof proteinsand nucleic acids (2,9,13,15). CI+ryct After electrophoresis, proteins are ONH *l* often visualized by staining the gel A "o *A*\., with (Fig- CoomassieBrilliant Blue 1 \Ar*.H'cre HOlS H ure 1). This dye has a high extinction coefficient in the visible spectrumupon complexation(16) and a high affinity UniblueA, 3 ProcionBlue MX-R,4 for proteins that does not depend strongly on the specific structures of the proteins(3). Other dyes (Figure 1), suchas FastGreen FCF 2 (l), Uniblue A 3 (5), ProcionBlue MX-R 4 (6) and T** Stains-all5 (8), are alsoused occasion- ally, although thesedyes are about ten @ times lesssensitive than dye 1 for stain- &1^ttA s02cl ing proteins. Stains-All,5 DansylChloride,6 Figure 1. Chemical structures of organic dyes 1{ used. Vol. 14.No. 6 (1993) BioTechniques925 Table l. Preparation of Solutionsof Dyes l-5 Used for Staining Protein Gels Concentration StainingSolution Dye (tlt|/vol) (vol/vol/vol) 1 0j% CHaOH/HzO/HOAc*(4:5: 1 ) 2 0.1"/" CzHsOH/HzOlHOAc(3:6: 1 ) 3 0.6% CHsOH/HzO/HOAc(5:4: 1 ) 4 0.05% CHsOH/HzO/HOAc(4:5: 1 ) 5 0.01% HCONHz/iPrOH/ Tris-HCl(15 mM, pH B.B)(2:5:13) *Ac = acetate MATERIALS AND METHODS Savin (Brighton, MA), Universal (UnitedStationers Supply. Des Plains, Chemicalsused for electrophoresis IL). Membranesused fbr nucleicacid were obtained from Bio-Rad (Hercu- and proteinblot were DEAE-cellulose les, CA). Organic dyes Coomassie (Schleicher& Schuell,Keene. NH), ni- Brilliant Blue 1. Fast Green FCF 2. trocellulose and Zeta-Probe(i'(Bio- Uniblue A 3 and dansyl chloride 6 Rad). were purchasedfrorn Sigma Chemical (St. Louis, MO). The Procion Blue Preparation of Bovine Serum Albu- min (BSA) Conjugatesof 3, 4 and 6 MX-R 4 andStains-all 5 wereobtained from Fluka (Ronkonkoma,NY) and To a solutionof BSA (100rri-s, 1.5 Aldrich (Milwaukee,WI), respective- mmol) in 0.1 M sodiumburate buffer ly. The transparencysheets were the (9 ml. pH 9.0)were added 3. 4 ur 6 ( l5 type intendedfbr use in plain-paper mmol) as the solid at room tempera- copiers.Eight commercialtransparen- ture. The reactionwas stimedat room cies were testedfor transf'erblotting: temperaturefbr 2 h and then subjected Arkwright PPC-IX (Arkwright, Fiske- to dialysisagainst water (2 x 4liters) ville, RI), ACP (American Coated for 48 h at 4oC. The dialyzed protein Products, Clinton, CT), Labelon solution was lyophilized.BSA-3 and (Canandaigua,NY), 3M PP2500(3M BSA-4 conjugateswere isolated as Visual SystemsDivision, Austin, TX), bluepowders and the B5,4-6conjugate Pitney-Bowes(Stamfbrd, CT), Polar- as a yellow powder.All proteinconju- oid 731 (Polaroid,Cambridge, MA), sateswere storedat 4"C. A B Figure 2. Comparison of PACE gels with the transfer blots onto transparencies.(A) An SDS- PAGE gel(l2c/c T lacrylamidel,0.37c C lcross-linkerl)ol mixtureso1'proteins stained u'ith Coomassic Blue 1. Lane l. serumalbumin fiom bovine,lane 2. proteinmol wt standards:(from bclttomto top) carbonicanhydrase (30000; from bovineerythrocyte). albumin (21-5000: frorn cgg), serumalbunrin (66000;from bovine),phosphorylase b (97400; fiom rabbitmuscle). B-galactosidase (116000: tiom E. coll), myosin(205000; fiom rabbit muscle);lane 3. oligomersof bovinccarbonic anhydrase cross- linked by dimethyl subserimidate.(B) Transf'erblot of A onto transparency. Vol. 14.No. 6 (1993) BioFeedback Gel Electrophoresis and Transfer color from the PAGE gel to the trans- was carriedout using an agarosematrix Blotting parencywas accompaniedby the trans- rather than polyacrylamide, the blot- fer of protein:when the part of the sur- ting workedas well as it did with SDS- Gel electrophoresiswas carried out face layer that containedthe blue band PAGE gel. These resultsindicate that following the Laemmli procedure for was scrapedas a powder from the sur- neither the gel matrix (polyacrylamide sodium dodecyl sulfate (SDS)-PAGE face of the transparencyand subjected or agarose)nor the type of electropho- (10) and the Margolis procedurefor to proteinanalysis (i.e., hydrolysis in 6 resis (denaturing, non-denaturingor non-denaturingPAGE (11).The proce- N HCI and treatmentwith ninhydrin), isoelectricfocusing) influence the pro- dure available in Hames and Rick- amino acids were detected.The dye tein blotting from the gel to the trans- woods was used for isoelectricfocus- alonedid not give a positiveninhydrin parency. ing (IEF)-PAGE (2). SDS-agarose test(14). The dye I appearedto decom- electrophoresiswas carriedout follow- poseunder the conditionsused to hy- Transparencies, Films and ing the manufacturer's procedure drolyzeproteins (6 N HCl. ll}"C, 24 Membranes (ProSieve@gels; FMC BioProducts, h): it becamecolorless and showeda Rockland, ME). Electrophoresisused negativeninhydrin test. The transferred We surveyeda number of commer- the Bio-Rad mini PROTEAN@II sys- Arkwright transparencvshowed about cial transparenciesused for plain-paper tem for denaturingand non-denaturing ten times strongerninhl'drin test than copiers,organic films commonly used PAGE and the Multiphor apparatus the transparencyalone lrirth protein for the backing supportsof transparen- (PharmaciaBiotech, Piscataway,NJ) carbonicanhydrase used as the exam- cies and filtration membranes, and for IEF-PAGE. After the electrophore- ple) underthe sameexperimental con- membranesused for nucleic acids and sis,the gelswere stainedwith dyes1-5 dition (i.e., samedimensions of films protein blottingsto determinethe gen- (Table1), andvolumes of ninhydrinreagent). The erality of the transferblotting (seeMa- The gels were destainedusing the Mylar@film and the transparenc-n.'fiom terialsand Methods). We foundthat the same solutions without dy.. The Pitney Bowes shor,reda negativenin- blotting membranesnitrocellulose, the destainedgels were removed from the hydrin test. When \rc resLrb.jc.ctedthe Zeta-Wobeand DEAE-celluloseworked destaining solution and immediately samples obtained b1' scrapinu blue asexpected, but that only two (theArk- placedbetween two sheetsof transpar- bandsfrom the transparencrto electro- wright PPC-X and Polaroid 731) out ency. Both sides of the transparency phoresis, the correspondinuprotern of eight transparenciestested trans- were testedfor blotting. Sincethis dif- bands were obsen'ed. although the ferred the dye-proteincomplexes from fusion blotting fiom the PAGE gel can bandswere blurred (ue useda silver the gel to the transparency.Both sides take place from either side of the gel, stainin theseexperiments ). of the transparencyaccepted proteins. this proceduregives two mirror images We werenot ableto electrophureti- Neither the uncoatedMylar film nor but otherwise indistinguishablerepli- cally blot dye-proteincomplcres fiom the cellulosefilm worked. cas of the original PAGE gel. The PAGE gels to transparencies.We pre- transfer was proceed allowed to at sume that the transparenciesare pore- Other Dyes room temperaturefor 2 h or 30 min at less and not conductivein an electric ,40"C.the
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