p roduct i nformation s heet for research use only. not for use in diagnostic or therapeutic procedures. mobitec gmbh, lotzestra? 22a, d-37083 g?ttingen, tel.: ++49 551-70 722- 0, fax: ++49 551-70 722-22 e-mail: info@mobitec.de or order@mobitec.de, internet: http://www.mobitec.de -galactosidase from escherichia coli . -galactosidase catalyzes the hydrolysis of -d-galactoside to galactose and alcohol. f7m: 1.0 mg -galactosidase immobilized on polyvinyl , 780 units immobilized per cr-column. 100 l of a 5% lactose solution is digested to over 80% in 60 minutes nr.14 storage buffer: 50 mm tris/hcl, ph 7.8 add dtt to the storagebuffer, final concentration: 1 mm dtt nr.64 reaction buffer: 50 mm phosphate, 1 mm mgcl 2 , ph 7.8 add -mercaptoethanol, to the reactionbuffer, final concentration: 10 mm nr.63 washing buffer: 50 mm phosphate, 1 m nacl, ph 7.8 protocol for more details see mobitec-crc-handbook. 1. dilute delivered buffers (at least 2 ml each) with sterile doubly distilled water. for 1 application you need 0.25 ml 10x reaction buffer and 2.25 ml doubly distilled water+ 10 mm -mercaptoethanol 0.4 ml 5x washing buffer and 1.6 ml doubly distilled water 0.2 ml 10x storage buffer and 1.8 ml doubly distilled water+ dtt (1 mm ) the substrate should be in reaction buffer 2. equilibrate the cr-column with 2 ml reaction buffer. fill 2 ml reaction buffer into a syringe, let the reaction buffer run through the column by gravity to the upper filter. in case the buffer runs very slowly, apply pressure by a syringe. 3. load substrate solution in reaction buffer. small volumes (< 80 l): spin the cr-column 5 seconds in a benchtop centrifuge (2000 rpm are sufficient). let the substrate solution enter the matrix material. larger volumes: let the substrate solution run through the column. flow-rate: up to 80 l/minute keep the substrate in the column for about 1 minute at room temperature. higher turn-over is obtained when the substrate is applied to the column again or incubated for longer times. 4. elute the product solution. small volumes (< 80 l): elute the product with 500 l reaction buffer. larger volumes: let the substrate run through the column and elute the residual product solution with 500 l reaction buffer. it does not harm the columns if they run dry. 5. wash the column with 2 ml washing buffer. 6. equilibrate the column with 2 ml storage buffer. store the column at 4 c. never freeze a cr-column! mobitec # A5101 02/10/2000 # A5101 i mmobilized -g alactosidase f7 m
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