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

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

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 transparencies 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 convectionblotting 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 protein and the associateddye transfer; however, 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 scanning 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 sandwichwas disassembled field. We surveyedfour other dyes2, 3, 4 and the transparencieswere air-dried When the transparencywas pre- and 5, which are used less frequently andstored. treatedwith reagents.such as the gel- than 1 to stain gels, for utility in this destaining solution, organic solvents blotting technique. We found that all (acetone,dimethylformamide and di- protein patterns formed by staining methyl sulfoxide)and protein-denatur- PAGE gels with dyes 2, 3 and 4 trans- RESULTS AND DISCUSSION ing agents(6 M guanidinehydrochlo- ferred faithfully to transparencies. ride, I7o SDS), transfer of the Dyes 1{ have two key features in Procedure Using CoomassieDye 1 protein-dyecomplexes to the transpar- common:all areanionic and hydropho- ency still occurred.When transparen- bic. The proteins stained by the cat- When a polyacrylamidegel contain- cieshad beenpretreated with hot acetic ionic dye 5, however,did not transfer. ing proteinswas stainedwith Coomas- acid, which separatesthe thin coating These dyes do not react chemically sie Brilliant Blue 1 and the gel was films on which the image forms from with proteins but only form non-cova- pressedagainst the transparencyfor 2h the structuralbody of the transparency lent complexes(4). Not surprisingly, at room temperatureor 30 min at 40oC, (the Mylar backing), transfer did not gels developed using other types of the blue bandscharacteristic of the pro- occur. procedures(e.g., staining with silver or tein-dye complexes transferredto the When the electrophoresiswas con- gold) did not transferduring blotting. transparency. Figure 2 shows repre- ducted in the non-denaturing buffer, sentative results obtained with mix- blotting transferred the protein-dye Modification of the tures of proteinsthat were separatedby complexes to the transparency. The Protein SDS-PAGE(10). At the usuallevel of proteins on an IEF-PAGE gel stained resolution, the transfer faithfully with 1 readily transferredto the trans- Protein only transferredfrom gel to reproducedthe original. The transferof parency.When protein electrophoresis transparencywhen the gel had been

928 BioTechniques Vol. 14,No. 6 (1993) BioFeedback

stained with dyes. The association substrates for use with gel scanning This researchwas suppottedby the Na- between dye and protein involves some densitometers. tional ScienceFoundation mder the Engi' combination of hydrophobic and elec- neerinS ResearchCenter Initiative to the trostatic interactions (4). In an effort to REFERENCES M.l.T. BiotechnologyProcess Engineering increase both effects, we carried out l.Allen,R.E., K,C. Mesak and P.K. McAllis- Center(Cooperotive Agreement CDR-88' PAGE on the covalently linked BSA ter. 1980.Staining protein in isoelectricfo- 03014).Y.-H. C. thanksRebecca Hung for Fa\rGreen Anal Bi{rhem for rea- her helpfulcommenrs about the work 4t its conjugatesof 3, 4 and 6 two H,Xt a-$J'. stages.Adiress correspondenceto sons. The BSA-3 and BSA-4 conju- zL, a". r-.. s. andA, chrambach.t98t. initial gates have greater capacity for both Anatyricaland prepararile gel elcctrofocus- G.M Whitesides. electrostatic(by the sulfonate groups) ing,p 157-187rtl B D. Hamesand D. Rick- ^rD^Fi"' hydrophobic(by the arenegroups) woods(Eds )' GelElectrophmesi' andinteraitions uno int.n,.ry ioroL'J andGeorge M' -.",r.a,mk[{n':hif i-'*i*}5''.K,- Irffiir:-ril" themselves.The ",i 854.-6 conjugateis ii. ,r," q"-ii"ii,i,.,or microsram morehydrophobic, due to theiniroduc- quanritiesorprorein util,,,ng pli"l'pi..,r D_epartm-en_toJ L.hemistry don of a ;aphthalenegroup, and is protein-dyebinding. Anal. Biochim "" /:248 Harvard University 254 fluorescent.we foundthit bolhBSA-4 Pll:'l'. 12 Oxford Street andBSA-5 conjugares wererransrerred nl'li*l^T.1.,1'B-ln3l: ,s,l::lYT Cambridge.MA 02I 38 fromthe gel tothi transparency. with- i["ilflH'!;:K:lili:"i!]* " " out additional staining with dyes in the s.Datyner,A. andE. Finnimor;. 1971. A new acidic medium. Qualitatively, staining stainingmethod for assav ofprotcin\ on polv- acrvlamidegels Anal Biochcm-t2:'15-55' these BSA-4 and BSA-5 conjugates , withcoomassie dye1 increaiedrhe"11iff"'"lli;3jlf;i';1"";,ilillll3l-' efficiency of transfer.The 85,4-6 con- duresfir quantitariveesrmari()n ot. proteins jugate did not hansfer alone, but the on etectrofhoretic\rrip\. Bi,'.him. Biophys. complex of the dye I and the 85A.-6 Acta7113't7'391 conjugatesuccessfully transferred. T Cershoni'J'M'1988 Prolein blotrins: a man ual. Methods Biochem. Anal. -l-l:l-58. lnese results tnat anlonlc suggest 8.creen,M'R., J'v' Pastewkaand A.c. Pea- sulfonategroups present in dyes asso- coct<,iq.i:. 6ine.enriatsraininS oj phospho- ciate electrostaticallywith proteins and proreinson polyacrylamide gelqirh a carionic that this associationfacilitates fiansfer carbocyaninedye. Anal. Biochcm. -56:43-51. q!Tl,."..o- polv- blofting. Hydrophobic interactions ap- i roductionto acnlamide qelln!l- electrophorc\r\. ln p. l-91. /, pear less rmponant ror lne Lranslerol e.6. Hames-and D. Ii,ck\roo.j{Ed\.). uel protein-dye complexes to tmnsparen- ElecrroDhoresisof proteins: A practicalAp- cies (41. Droach.IRL Press.Oxford. l0.Laemmli, U.K. 1970.Clcavage of structural proteinsduring the assembll ot' the head of Advantages bacteriophageT4. Nature227:680-685. ll.Margolis, J. and K.G. Kendrick. 1968. We have described a convenient Polyacrylamidegel electrophoresisin a con- method for long-term, non-photo- tinuous molecularsieve gradicnt.Anal. Bio- graphic storageof the information pre- chem.25:341-362. sent in electrophoresisgels, basedon l2.Pluskal, M.G., M.B. Przekop, M.R. Ka- vonian. C. Vecoli and D.A. Hicks. 1986.Im- blotting protein patterns,after staining mobilon PVDF transfer membrane:a new with Coomassie Brilliant Blue and membranesubstrate for Western blotting of other dyes, onto commercial trans- proteins.BioTechniqu es 1:212-282. parencies. These transparencies are l3.Rickwood, D. and B.D. Hames. 1990.Gel readily availableand inexpensive.The Electrophoresisof Nucleic Acids: A Practical Approach,2nd Edition. IRL Press,Oxfbrd. blotted transparenciesretain the infor- l4.Sameijima, K., W. Dairman and S. Uden- mation presentin the originally stained friend. 1971.Condensation of ninhydrrnwith gel. They are dimensionallystable, me- aldehydesand primary aminesto yield highly chanically sturdy, flat and convenient fluorescentternary products.L Studieson the to store. This method provides a sim- mechanismof the reaction and some charac- teristics of the condensation product. Anal. ple, convenientand inexpensive way to Biochem.42:222-236. preservedata from protein electropho- l5.Takeo, K. 1987.Affinity electrophoresis,p. resis and an effective method of dis- 229-2'79.1n A. Chrambach,M.J. Dunn and playing data using an overheadprojec- B.J.Radola (Eds.), Advances in Electrophore- tor (in particular, the patterns in the sis,Volume l. VCH Publishers,New York. l6.Tal. M.. A. Silberstainand E. Nusser. 1985. transparency preserved their original Why doesCoomassie Brilliant Blue R interact colors on transfer). These dye-stained differently with different proteins? A partial transparencies are also excellent answer.J. Biol. Chem. 260:9916-9980.

930 BioTechniques