Precast Gel CriterionXT Instruction Guide - Bio-Rad

1 Criterion XT Precast GelInstruction GuideFor Technical Service Call Your Local Bio-Rad Office or in the US, Call 1-800-4 BIORAD(1-800-424-6723)CCaattaalloogg 3/25/2003 2:19 PM Page iTable of ContentsSection 1 General Criterion XT Precast Criterion System Criterion XT Comb 4 Section 2 Setup and Basic Setting Up and Running Criterion XT Opening Criterion XT Cassettes and Removing the 3 SDS-PAGE and Native Criterion XT Gel Selection Bis-Tris Gel Tris-Acetate Gel Criterion XT Buffers and Sample Running Conditions .. 3/25/2003 2:19 PM Page iSection 4 2-D Agarose Overlay .. 11 Section 5 Staining and SDS-PAGE and Native PAGE 13 Section 6 14 Section 7 16 Section 8 Ordering 17 Criterion XT Criterion XT Buffers and Kits.

2 Other Related Criterion Criterion Gel Protein Standards .. Detection Blotting 3/25/2003 2:19 PM Page iiSection 1 General is the next generation of dedicated Precast gel systems. The innovative, easy-to-use designproduces superior resolution while allowing you to run more samples per gel. Compared to any other precastgel system, Criterion produces more results while providing significant cost and time savings. Some of theunique features and benefits provided are: 12 month shelf life for Bis-Tris gels 8 month shelf life for Tris-acetate gels Room temperature storage for Bis-Tris gels Easy sample preparation without extra anti-oxidant addition steps Patented integral buffer chamber that eliminates buffer leaks Up to 26 sample capacity per gel Flexibility to run one or two gels Multichannel pipet compatible gels Outlined and numbered wells that simplify sample loading J-foot that improves gel drying and blotting resultsUS Patents #5,073,246, #5,656,145, #6,093,301 and other patents issued and 3/25/2003 2.

3 19 PM Page XT Precast GelsCriterion XT Precast gels are formulated at pH near neutrality to optimize gel matrix stability, significantlydelaying acrylamide hydrolysis, which occurs in traditional Laemmli systems. Specially optimized buffersresult in tight, consistently resolved bands throughout the life of the versatile system allows the separation of small to large proteins using just two gel buffer systems:Criterion XT Bis-Tris Precast gels for small to mid-sized proteins and Criterion XT Tris-acetate Precast gels forlarge Criterion XT Bis-Tris gels are based on a buffer system (pH ) that uses discontinuouschloride and MES or MOPS ion fronts to form moving boundaries to stack and then separate denaturedproteins by size.

4 The chemistry of the XT Bis-Tris gels allows maximum stability and consistent results for aminimum of one year. Running the same XT Bis-Tris gels with the XT MES denaturing running buffer or the XTMOPS denaturing running buffer will produce different migration patterns. A combination of these two runningbuffers and our three XT Bis-Tris gels can produce up to six different migration patterns in the small and mid-size Criterion XT Tris-acetate gels are based on a Tris-acetate buffer system (pH ). It uses discontinuousacetate and Tricine ion fronts to form moving boundaries to stack and then separate large denatured proteinsby molecular weight. The Criterion XT Tris-acetate gels can also be used to separate proteins by their charge-to-mass ratio (under native-PAGE conditions).

5 This is possible because the XT Tris-acetate gels are madewithout SDS, allowing the sample buffer and running buffer to dictate the separation mechanism. Thenonreducing and nondenaturing environment of native PAGE allows the detection of biological activity andcan improve antibody detection. Native PAGE can also be used to resolve multi-protein bands wheremolecular mass separation by SDS-PAGE would reveal only one and for the separation of intact 3/25/2003 2:19 PM Page 2complexes. Separation by native PAGE with XT Tris-acetate gels uses discontinuous acetate and glycineion fronts to form moving boundaries to stack and separate proteins by both size and samples for the Criterion XT Precast gel system are prepared in a reducing denaturing sample sample buffer contains XT reducing agent, a pH neutralized and stabilized solution of TCEP as thereducing agent; heat and SDS are used to denature the proteins.

6 In addition, the use of TCEP in combinationwith Bio-Rad s optimized running buffers maintains proteins in a fully reduced state during the electrophoresisrun, eliminating the need for an anti-oxidant in the upper buffer chamber. Criterion XT Tris-acetate precastgels can also be used for native PAGE. Proteins are prepared in a nonreducing, nondenaturing sample buffer,which maintains the proteins native structure and charge 3/25/2003 2:19 PM Page System SpecificationsGel materialPolyacrylamideGel dimensions (W x L) x cmGel mmResolving gel cmCassette dimensions (W x L) x cmCassette materialStyrene copolymer Comb materialPolycarbonate Storage tray materialPET Upper running buffer volume60 mlLower running buffer volume800 mlStorage conditionsBis-Tris gels: Store flat at ambient temperature; DO NOT FREEZETris-acetate gels: Store flat at 4 C; DO NOT FREEZEGel shelf life12 months for Bis-Tris gels.

7 8 months for Tris-acetate Criterion XT Comb ConfigurationsCombLoad VolumeComments12+2 well45 l with two 15 l reference wellsMultichannel pipet compatible18-well30 l 26-well15 l Multichannel pipet compatiblePrep+2 well800 l with two 15 l reference wellsIPG11 cm ReadyStrip IPG stripIPG+1 well11 cm ReadyStrip IPG strip with one 15 l reference 3/25/2003 2:19 PM Page 4 Section 2 Setup and Basic Up and Running Criterion XT Gels1. Each Criterion XT gel is packaged individually in a plastic storage tray. Remove the cover by gently pullingthe corner tab up and diagonally across the package. Remove the gel from the Remove the comb and gently rinse the wells with ddH2O or running Remove the tape from the bottom of the cassette by pulling thetab across the Insert the Criterion XT gel into one of the slots in the Criterion celltank.

8 Ensure that each integral buffer chamber faces the center ofthe cell. 5. Fill each integral buffer chamber with 60 ml running Load samples using a Hamilton syringe or a pipet with gel loadingtips. A sample loading Guide can be placed on the outer edge ofthe cassette to aid in aligning pipet tips with the wells. This isespecially useful with multichannel Fill each half of the lower buffer tank with 400 ml of running bufferto the marked fill 3/25/2003 2:19 PM Page 58. Place the lid on the tank, aligning the color-coded banana plugs and jacks. See section for Criterion XT Cassettes and Removing the Gels1. After electrophoresis is complete, turn off the power supplyand disconnect the electrical Remove the lid from the tank and remove the Criterion XTgel(s) from the cell.

9 Pour off and discard the upper Invert the cassette and place the integral buffer chamber overthe cassette-opening tool built into the Criterion cell Firmly press down on the cassette to crack the cassette weldson both sides of the cassette. The cassette will split openapproximately 1/3 of the Alternatively, the gel cassette can be opened by sliding thetapered back of the comb into the slits on either side of Pull the two halves of the cassette apart to completely exposethe Remove the gel by either floating the gel into a fixing or stainingsolution or by carefully lifting the gel from the 3/25/2003 2:19 PM Page 6 Section 3 SDS-PAGE and Native XT Gel Selection GuideCriterion XT gels are available in a wide selection of single acrylamide percentages and gradients for theseparation of proteins by SDS-PAGE or native SeparationBis-Tris GelsWith XT MES Running Buffer With XT MOPS Running Buffer10% 200 kD14 220 kD12%1 30 kD 6 66 kD4 12% 200 kD10 300 kD Tris-Acetate Gels*With XT Tricine Running BufferWith Tris/Glycine Running Buffer7%36 200 kD N/A3 8%40 400 kD N/A*Because Criterion XT Tris-acetate gels are made without SDS, they can be used to separate proteins by both SDS-PAGE and native 3/25/2003 2.

10 19 PM Page 7815025010075503725202501501007550372520 1510250150100755037252015250150100755037 2501501007550372501501007550372520151015 0250100755037252015101502501007550 372520151010%12%4 12%10%12%4 12%7%3 8%Bis-Tris gels withXT MES running buffer:ideal for SMALL proteinsBis-Tris gels withXT MOPS running buffer:ideal for MID-SIZE proteinsTris-acetate gels withXT Tricine running buffer:ideal for LARGE proteinsCriterion XT Protein Migration 3/25/2003 2:19 PM Page Gel CompositionGel , pH linker5% CStacking gel4% T, 5% CStorage , pH life12 months; individual expiration date is printed on each cassette; store flat at ambient Gel CompositionGel bufferTris-acetate, pH CStacking gel4% T, CStorage bufferTris-acetate, pH life8 months.