Immobiline® DryStrip Reswelling Tray User Manual
Contents
1. M aintenance section Refer to the Immobiline DryStrip Kit instructions for a complete guide to handling Immobiline DryStrip gels Immobiline DryStrip Gel Rehydration Rehydration solution volume Ideally the rehydration solution volume for an Immobiline DryStrip gel should equal the amount of fluid removed during the drying process This ensures that the entire sample if included in the solution enters the gel It also minimizes electroendosmotic flow and water exudation dur ing the focusing step Immobiline DryStrip length Volume per slot 11 cm 200 240 ul 18 cm 340 400 ul Rehydration solution The final concentration of IPG Buffer used for rehydration of IPG strips depends on the type of second dimension gel electrophoresis used To run a horizontal second dimension use the information listed in Table 1 and to run a vertical second dimension use the information list ed in Table 2 Note The rehydration solution can be stored in aliquots at 20 C Table 1 Horizontal Second Dimension Component Final Immobiline DryStrip type conc 3 10L 3 10NL 4 7L Urea 8 M 24g 24g 24g CHAPS 2 0 1g 0 1g 0 1g IPG Buffer pH 3 10L 0 5 25 ul IPG Buffer pH 3 10NL 0 5 25 ul IPG Buffer pH 4 7L 0 5 25 ul Dithiothreitrol DTT 0 3 15 mg 15 mg 15 mg Bromophenol Blue trace add just a few grains Double distilled water to 5 ml to 5 ml to 5 ml Table 2 Vertical Second Dimension Component Final2. Immobiline DryStrip type conc 3 10L 3 10NL 4 7L Urea 8 M 24g 24g 24g CHAPS 2 0 1g 0 1 g 0 1g IPG Buffer pH 3 10L 2 0 100 ul IPG Buffer pH 3 10NL 2 0 100 ul IPG Buffer pH 4 7L 2 0 100 ul Dithiothreitrol DTT 0 3 15 mg 15 mg 15 mg Bromophenol Blue trace add just a few grains Double distilled water to 5 ml to 5 ml to 5 ml 3jf necessary the concentration of urea can be increased to 9 or 9 8M Other non ionic detergents like Triton X 100 NP 40 or n octyl B glucoside can be used Orange G can be used instead of Bromophenol Blue Sample capacity The protein capacity of Immobiline DryStrip gels ranges from several ug to mg Factors that determine the maximum amount of sample for a strip include protein solubility and the combination of pH gradient width and strip length In general the longer the strip and the narrower the pH gradient the higher the protein capacity will be Rehydration protocol Day 1 The rehydration protocol applies to both simple reswelling with later application of the sample by the cup method and reswelling with sam ple To proceed the appropriate rehydration solution should already be prepared with or without sample 1 Slidethe protective lid completely off of the reswelling tray 2 Level the reswelling tray by turning the levelling feet until the bubble in the spirit level is centered 3 Pipette the appropriate volume of rehydration solution into each slot N
3. L 12 17 1234 01 pH 3 10 NL 12 17 1235 01 PlusOne Reagents Urea 500 g 17 1319 01 Triton X 100 500 ml 17 1315 01 CHAPS 1g 17 1314 01 Dithiothreitol DTT 1g 17 1318 01 Bromophenol Blue 10g 17 1329 01 DryStrip Cover Fluid 1 liter 17 1335 01 IPG Buffer for IEF IPG Buffer pH 4 7L 1 ml 17 6000 86 IPG Buffer pH 3 10L 1 ml 17 6000 87 IPG Buffer pH 3 10NL 1 ml 17 6000 88 Printed in the USA
4. Immobiline DryStrip Reswelling Tray User Manual e Pharmacia 80 6375 64 Rev B 5 97 Biotech English Important user information Please read this entire manual to fully under stand the safe and effective use of this prod uct Should you have any comments on this manu al we will be pleased to receive them at Hoefer Pharmacia Biotech Inc M arketing Department 654 M innesota Street San Francisco CA 94107 3027 Pharmacia Biotech AB reserves the right to make changes in the specifications without prior notice Warranty and Liability Pharmacia Biotech guarantees that the prod uct delivered has been thoroughly tested to ensure that it meets its published specifica tions The warranty included in the condi tions of delivery is valid only if the product has been installed and used according to the instructions supplied by Pharmacia Biotech Pharmacia Biotech shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Copyright 1997 Pharmacia Biotech AB All rights reserved No part of this publica tion may be reproduced stored in a retrieval system or transmitted in any form by any means without permission in written form from the company Fran ais Renseignements important
5. mmobiline DryStrip Kit Instructions and in 2 D Electrophoresis Application N otes available from Pharmacia Biotech Note Complex protein samples containing 200 500 ug protein or more generally require a longer focusing period 60 100 kVh A slow voltage ramp for the first 3 to 6 hours may improve focusing and decrease sam ple aggregation Care and Maintenance N ever autoclave or heat any part above 45 C After each use rinse the reswelling tray thoroughly and treat with a detergent solution to remove residual protein Rinse thoroughly with water and then double distilled water and allow to dry N ever clean the reswelling tray with alcohols organic solvents abra sives strong cleaning solutions or strong acids or bases Customer Service Information Technical Service and Repair Pharmacia Biotech offers complete technical support for all our prod ucts If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Pharmacia Biotech representative Important Request a copy of the Pharmacia Biotech H ealth and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this form is properly completed Ordering Information Qty Code No Immobiline DryStrip Reswelling Tray 1 80 6371 84 Immobiline DryStrip Gels 11cm pH 4 7 L 12 18 1016 60 pH 3 10 L 12 18 1016 61 18 cm pH 4 7 L 12 17 1233 01 pH 3 10
6. ote Deliver the solution slowly carefully and at one point to minimize the introduction of air bubbles Remove any larger bubbles that may be trapped in the solution 4 Gently lay one Immobiline DryStrip gel gel facing down on the solution in each prepared slot Note For ease of removal after rehy dration place the strip so that the pointed end abuts the sloped end of the slot To remove a strip slide the forceps tip along the slope and into the slight depression under the strip 5 Overlay each Immobiline DryStrip gel with 2 3 ml of DryStrip Cover Fluid to prevent evaporation and urea crystallization 6 Slide the lid onto the reswelling tray and allow the gels to hydrate overnight at room temperature Day 2 1 Lift each strip and gently blot the plastic backing of each strip with filter paper to remove excess moisture and oil 2 If the rehydration solution included the sample place each strip into the M ultiphor II and proceed skipping any instructions pertaining to sample cups in the Immobiline DryStrip Kit instructions To apply a sample using sample cups refer to the appropriate section in the Immobiline DryStrip Kit instructions Place one DryStrip Gel per Spirit level f N reswelling chamber up to 12 total Levelling feet 2 Electrophoresis Follow your selected protocol or follow the guidelines for first dimen sion focusing and second dimension gel electrophoresis that can be found in the I
7. s d utilization Pour une bonne compr hension et une utilisation en s curit maximale il convient delire enti rement ce manuel Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser Hoefer Pharmacia Biotech Inc M arketing Department 654 M innesota Street San Francisco CA 94107 USA Pharmacia Biotech se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Garantie et responsabilit Pharmacia Biotech garantit l utilisateur que le produit livr a subi avec succ s tous les essais pr vus pour s assurer qu il est conforme aux sp ci fications et normes en vigueur La garantie incluse dans les conditions de livraison n est valable que si le produit a t install et utilis conform ment aux instructions fournies par Pharmacia Biotech La soci t Pharmacia Biotech ne sera en aucun cas responsable de tout dommage caus directement ou indirectement par toute utilisation incorrecte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes toute perte de perspectives commerciales tout emp chement d utilisation et tout autre risques ayant un rapport avec l utilisation du produit mais sans aucune limitation quant la nature de ces dommages Copyright 1997 Pharmacia Biotech AB Tous droits r serv s La reproduction le stockage dans un syst me de r cup ration d informations o
8. u la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la pr sente publi cation en totalit ou en partie sont strictement interdits sans autorisation pr alable crite de la soci t Function and Description The Immobiline DryStrip Reswelling Tray is an accessory to the M ultiphor II and the Immobiline DryStrip system for 2 D electrophore sis The tray has twelve independent reservoir slots that can each hold one Immobiline DryStrip gel up to 18 cm long Separate slots allow the rehydration of individual Immobiline DryStrip gels in a minimal volume of solution see Sanchez J C et al Electrophoresis 18 1997 By including the sample to be separated in the rehydration solution the sample does not need to be loaded subse quently through the conventional sample cup Loading the sample by rehydration eliminates precipitation at the application site and increases the amount of sample that can be loaded to 5 mg or more The tray includes a lid to protect the strips from dust and contamination during the rehydration period which may range from 6 hours to overnight Leveling feet and an attached spirit level aid in the even appli cation of solution Important notes Always wear gloves when handling Immobiline DryStrip gels Do not overfill the slots the proper rehydration volume is tabulated in the following section Clean the reswelling tray thoroughly after each use see the Care and
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