Agilent 3100 OFFGEL Fractionator Kit
Contents
1. Procedures To prepare solutions and sample for Peptide Fractionation 1 Prepare Peptide OFFGEL Stock Solution 1 25X a Add the components in Table 6 to an empty 50 mL container Table6 Peptide OFFGEL Stock Solution 1 25X Tube Amount Comment Glycerol Solution 6 mL OFFGEL Buffer can be 600 pL Choose the appropriate omitted at the cost of ampholytes depending some loss of resolution if on the IPG strip used downstream analysis pH 3 10 or 4 7 shows interference with ampholytes b Mix contents and bring total volume to the 50 mL mark on the tube with dH 0 c Label the 50 mL tube depending on which ampholytes are used You can aliquot the OFFGEL Stock Solution into smaller tubes to avoid multiple freeze thaw cycles OFFGEL Stock Solution can be stored at 20 C for four months 2 Prepare Peptide IPG Strip Rehydration Solution Refer to Table 7 for a single sample Table 7 Peptide IPG Strip Rehydration Solution 24cm strip 12cm strip 24 well frame 12 well frame Peptide OFFGEL Stock Solution 1 25X 0 96 mL 0 56 mL dH70 0 24 mL 0 14 mL Total Solution 1 2 mL 0 7mL 8 OFFGEL Fractionator Kit User Guide OFFGEL Fractionator Kit User Guide Procedures If you are using a multichannel pipettor you will want to make extra IPG Strip Rehydration Solution so there is excess in the reservoir tray 3 Prepare OFFGEL Peptide Sample For each sample to be prepared add the OFFGEL Stock Solutio2. Every 24 hours of operation exchange the upper electrode pads with fresh pads wetted in deionized water a Open the lid this will pause the fractionation b Remove the movable electrode c Carefully slide the tray to the right d Remove the fixed electrode without lifting the tray from the platform e Exchange the pads using tweezers f Refill cover fluid if needed some creeping of cover fluid to empty neighboring lanes is normal and does not impact fractionation quality Do not just reapply OFFGEL solution or water to electrode pads during the fractionation The liquid can seep between tray and IPG strip and current can bypass the sample 16 OFFGEL Fractionator Kit User Guide Procedures Best Practices If you are not using all lanes start with loading the samples from the middle lanes of the tray outward Ifsample is leaking out of the frame remove the sample with a pipette the sample may be used again remove the frame clean and dry the tray and start over using a new IPG strip Sample leakage can be avoided by making sure the frame is snapped onto the tray tightly and the IPG strip is rehydrated completely e The upper electrode pads needs to be exchanged with fresh electrode pads wetted in deionized water every 24 hours e Cover fluid may be reapplied during the fractionation but should not be added to a higher level than 1 2 height of the tray groove Typical starting voltages depending on con
3. Kit pH 4 7 3100 OFFGEL Starter Kit OFFGEL Protein Test Sample 3100 OFFGEL 12 Well Frame Set 3100 OFFGEL 24 Well Frame Set Tray Set includes 4 replacement trays Electrode Assy includes 1 tray 1 fixed electrode and one moveable electrode USB Memory Stick Kit includes 256 MB USB flash memory Literature Reference 1 A Rapid Method to Desalt and Concentrate Proteomic Samples Using the Agilent mRP C18 Column Agilent Publication Number 5989 2506EN 2005 Troubleshooting For troubleshooting information refer to Section 6 of the Agilent 3100 User Manual 22 OFFGEL Fractionator Kit User Guide Notes Notes OFFGEL Fractionator Kit User Guide 23 www agilent com Agilent Technologies Inc 2006 2010 Printed in Germany Fifth Edition September 2010 G3100 90113 if Agilent Technologies
4. manual in section 4 page 65 Performing a fractionation To retrieve the fractions After fractionation be careful when you remove the cover seals Do not lift the well frames or contaminate the fractions with cover fluid e Use a pipette or a multipipettor to carefully pipette the Sample Solution from the frame wells while avoiding aspirating the IPG gel Please see the User Guide Section 4 for details on how to export results To run a test sample OFFGEL Fractionator Kit User Guide Beta lactoglobulin Agilent part number 5188 6428 OFFGEL Protein Test Sample can be used to evaluate the performance of the system 1 Prepare the beta lactoglobulin Test Sample Stock by weighing 1 0 mg and placing it in an Eppendorf or other appropriate container with 1 0 mL of dH 0 This Test Sample Stock can then be used to make a Prepared OFFGEL Sample according to the directions in Prepare the OFFGEL Protein Sample on page 6 Recommended amount of the Protein Test Sample is 50 ug Combine the components in the table below to prepare the Test Sample Procedures Table 9 Test Sample Preparation 24 cm strip 12 cm strip 24 well frame 12 well frame Test Sample Stock 50 uL 50 uL dH70 670 uL 310 uL Protein OFFGEL Stock Solution 2 88 mL 1 44 mL 3 Fractionate this Test Sample as you would a Prepared OFFGEL Sample using either the 24 well or 12 well default methods 0G24PR00 or 0G12PR00 4 Ifthe inst
5. Agilent 3100 OFFGEL Fractionator Kit Quick Start Guide Before you begin 2 Reagent Kit Checklist 2 Required Equipment and Reagents 2 Sample and Stock Solution Preparation Notes 3 Procedures 5 To prepare solutions and sample for Protein Fractionation 5 To prepare solutions and sample for Peptide Fractionation 8 To assemble the IPG strips frames and electrodes 10 Best Practices 17 To start a fractionation run 19 To retrieve the fractions 19 To run a testsample 19 Reference 22 Product Information 22 Literature Reference 22 Troubleshooting 22 OFFGEL Electrophoresis is a novel technique that separates proteins or peptides according to their isoelectric points whereby the separated components are recovered in liquid fractions Downstream sample processing steps like protein digestion and liquid chromatography can be easily interfaced with this method for multi dimensional separations of complex samples This guide describes the proper use of the reagent kits with the Agilent 3100 OFFGEL Fractionator phe Agilent Technologies Before you begin Before you begin Reagent Kit Checklist IPG Strips store at 20 C Frames Cover Seal Urea in 50 mL bottle 25 2 g electrophoresis grade Thiourea 9 1 g analytical grade DTT dithiothreitol 600 mg electrophoresis grade store at 4 C Glycerol Solution OFFGEL buffer containing ampholytes store at 4 C Electrode Pads Mineral Oil Cover Fluid Required Equipment and Reag
6. Ampholytes Ampholytes can interfere with MS ionization The amount of ampholytes can either be reduced to 0 25 a quarter of the standard concentration or they can be omitted completely in the OFFGEL solution This will lead to a slight decrease of resolution Also ampholytes can easily be removed if the sample is cleaned with reverse phase LC using a prior trapping column For better visibility during rehydration bromphenol blue may be added to rehydration or OFFGEL solution at a concentration of 0 002 Fractionation times and performance are not impacted by bromphenol blue e In case of low liquid levels in external wells when fractionating proteins it is normal for external wells to have reduced liquid levels after fractionation Proteins and DTT carrying a charge in the alkaline region drag water which leads to wells with lower protein concentration to be lower in liquid OFFGEL solution without ampholytes can be refilled in those wells during the run Also the glycerol concentration can be increased to 10 to increase the viscosity of the solution It may help to either reduce the DTT concentration or to avoid the DTT altogether by treating the sample with iodoacetamide and alkylation prior to OFFGEL 18 OFFGEL Fractionator Kit User Guide Procedures To start a fractionation run e Please see the User Guide Section 4 for operation of the 3100 OFFGEL including starting a run A list of available methods is shown in the
7. OFFGEL Fractionator Kit User Guide Procedures Procedures To prepare solutions and sample for Protein Fractionation WARNING Wear hand and eye protection and follow good laboratory practices when you prepare and handle reagents and samples 1 Prepare Protein OFFGEL Stock Solution 1 25X a Locate the bottle labelled Urea Add the following components to the Urea bottle Table 1 Protein OFFGEL Stock Solution 1 25X Comment Tube Amount Thiourea Entire tube DTT Entire tube Glycerol Solution 6 mL OFFGEL Buffer 600 uL Rinse with dH 0 if needed Rinse with dH 0 if needed Choose the appropriate ampholytes depending on the IPG strip used pH 3 10 or 4 7 b Mix the contents and bring the total volume to the 50 mL mark on the bottle with dH 0 You may need to warm the solution in warm water to dissolve OFFGEL Fractionator Kit User Guide Procedures CAUTION Do not let water temperature exceed 37 C Elevated temperatures cause urea to hydrolyze to isocyanate which modifies proteins by carbamylation c Label the 50 mL bottle depending on which ampholytes are used You can aliquot the OFFGEL Stock Solution into smaller tubes to avoid multiple freeze thaw cycles OFFGEL Stock Solution can be stored at 20 C for four months 2 Prepare Protein IPG Strip Rehydration Solution Follow the table below for a single sample Table 2 Protein IPG Strip Rehydration Solution 24 cm stri
8. centration of sample and salt content are 200 to 1500V If the starting voltage is above 1500 V there may be a contact issue electrode does not contact electrode pad IPG strip If starting voltage is below 100 V the salt concentration of the sample may be too high To improve poorly resolving protein samples precede the standard method with a ramp up phase to allow slow sample entry into the gel e g 300 V for 2 h followed by 600 V and 1500 V for 2 hours each See the User Manual for modification of methods e To eliminate leakage and cross contamination between samples do not reuse frames and seals See part numbers 5188 8012 and 5188 8013 OFFGEL Fractionator Kit User Guide 17 Procedures e Remove additives after fractionation Salt urea Desalt OFFGEL protein fractions if they are to be digested or analyzed directly by MS To desalt use mRP Agilent or Zeba columns Pierce This step removes urea thiourea and glycerol Glycerol Glycerol in peptide fractions can interfere with speed vac concentration increased viscosity direct injection into reverse phase LC or with crystallization ona MALDI target Glycerol can be removed by dialysis LC trapping columns mRP Agilent or C18 ZipTips Millipore You can also omit glycerol from the OFFGEL solution This may lead to reduced liquid levels in some wells These wells can be refilled every few hours of fractionation with ampholyte and glycerol free OFFGEL solution
9. e upper pads should be replaced every 24 hours of fractionation see step 23 14 Wait 15 minutes to allow the IPG gel to swell 15 After allowing 15 minutes for reswelling of the gel load 150 uL of Prepared Protein or Peptide OFFGEL sample OFFGEL stock solution Sample into each well e 24cm strip 24 well frame 150 uL X 24 wells 3600 uL e 12 cm strip 12 well frame 150 uL X 12 wells 1800 uL OFFGEL Fractionator Kit User Guide 13 Procedures To assemble the IPG strips frames and electrodes z 16 Place the cover seal over the frame and press down gently on each well to secure proper fit Be careful not to move the frame in the tray 17 Reapply 10 uL of dH 0 onto the electrode pads at each of the IPG gel ends Be careful not to move the pads 18 Place the tray on the instrument platform 14 OFFGEL Fractionator Kit User Guide Procedures To assemble the IPG strips frames and electrodes 19 Pipette cover fluid mineral oil onto the gel strip ends in several steps When pipetting cover fluid use caution not to move the electrode pads For 24 well frames a Pipette 200 uL cover fluid onto the anode end fixed electrode of the IPG strip Pipette 400 uL cover fluid at the cathode side movable electrode b After 1 minute reapply an additional 200 uL cover fluid to both ends of the IPG strip c After 3 minutes add an additional 200 uL cover fluid onto the anode end fixed electrode of t
10. ents 3100 OFFGEL Fractionator OFFGEL Tray OFFGEL Electrodes dH 0 Adjustable Pipettors 100 uL 200 uL amp 1000uL also 20 uL amp 200 uL adjustable multichannel pipettors recommended 50 mL conical tube for making Peptide OFFGEL Stock Solution 5 to 15 mL conical tubes for Prepared OFFGEL Samples Tweezers Reservoir trays for multichannel pipettors OFFGEL Fractionator Kit User Guide Before you begin Sample and Stock Solution Preparation Notes OFFGEL Fractionator Kit User Guide Always wear laboratory gloves when handling and preparing solutions samples apparatus etc to prevent contamination of samples and solutions i e skin keratin The maximum protein load will vary based on the complexity and solubility of each sample For complex samples such as serum plasma cell lysate and tissue samples we recommend a range of 50 ug to 5 mg For purified single proteins or simple mixtures the maximum capacity will be less than that for complex samples Substances that can interfere with IEF separation such as salts should be avoided Total salt concentration in the sample should not exceed 10 mM for optimal fractionation Samples can be desalted via dialysis mRP desalting ref 1 or buffer exchanged into a low salt buffer such as 10 mM Tris HCL pH 7 6 The sample should not be in a buffer with greater than 10 mM concentration Non ionic or zwitterionic detergents like CHAPS can be used in OFFGEL electropho
11. he IPG strip The cover fluid should not extend higher than 1 2 the height of the tray grooves For 12 well frames a Pipette 200 uL cover fluid onto the anode end fixed electrode of the IPG strip Pipette 1 mL cover fluid at the cathode side movable electrode b After 1 minute reapply an additional 200 uL cover fluid to both ends of the IPG strip The cover fluid should not extend higher than 1 2 the height of the tray grooves 20 Select the fixed electrode Anode see Figure 4 in Section 1 of the Agilent 3100 User Manual and place the two tabs on the electrode into the slots on the left side of the tray Rotate the fixed electrode down into position over the electrode pad insert Push down until the fixed electrode clicks into place Slide the tray without lifting it into the anode connector OFFGEL Fractionator Kit User Guide 15 Procedures To assemble the IPG strips frames and electrodes 21 Insert the moveable electrode Cathode Figure 4 in Section 1 of the Agilent 3100 User Manual in the orientation shown and push down until it clicks in place Lane labels on the electrode should read A H on the right side with A at the top of the electrode The movable electrode should touch the end of the frame Be sure that the moveable electrode makes contact with the electrode pad insert If not gently reposition the electrode pad 22 Close the lid and start the fractionation r gt 23
12. he end of the tray Be careful not to move or crimp the strip while placing the frame down 6 Lower the right side of the frame and press the frame down until it snaps in OFFGEL Fractionator Kit User Guide 11 Procedures To assemble the IPG strips frames and electrodes 7 Pipette 40 uL of IPG Strip Rehydration Solution into each of the wells Be careful not to touch the gel If you are using a multichannel pipettor prepare a larger batch of IPG Strip Rehydration Solution Multichannel pipettors are very useful for this step 8 After pipetting gently tap the tray onto the desk to ensure that the IPG Strip Rehydration Solution reaches the gel 9 Visually inspect to determine that the IPG Strip Rehydration Solution has wetted the gel surfaces The IPG Strip Rehydration Solution does not need to completely cover the exposed gel in the wells 10 From the container labelled Electrode Pads remove four pads for each IPG strip 11 Using tweezers wet one electrode pad with IPG Strip Rehydration Solution 12 Place one wetted electrode pad on each protruding end of the IPG strip Make sure that there is no gap between the pad and the frame 12 OFFGEL Fractionator Kit User Guide Procedures To assemble the IPG strips frames and electrodes 13 Place a second wetted electrode pad on top of the first electrode pad Each end of IPG strip should now be covered by 2 pads each which are on top of each other Th
13. n 1 25X to each sample in the amount indicated in Table 8 Table 8 OFFGEL Peptide Sample 24 cm strip 12 cm strip 24 well frame 12 well frame Peptide OFFGEL Stock Solution 1 25X 2 88 mL 1 44 mL sample 0 72 mL 0 36 mL The final salt concentration in the diluted sample should not exceed 10 mM If you will be running the same sample in multiple lanes you can use Table 4 and Table 5 to make multiple batches of the sample Procedures To assemble the IPG strips frames and electrodes To assemble the IPG strips frames and electrodes 1 Place tray in orientation shown with fixed electrode side on the left and tray handle on the right see Figure 4 in Section 1 of the Agilent 3100 User Manual 2 Remove the protective backing from the IPG strip gel Place the IPG strip in the tray with the gel side up Observe correct tray orientation with handle on the right and fixed electrode slots on the left Pull the strip as far left as possible until the strip touches the left edge of the tray Low pH side Anode marked with a must be at the left side where the fixed electrode will be attached See Figure 4 in Section 1 of the Agilent 3100 User Manual 10 OFFGEL Fractionator Kit User Guide Procedures To assemble the IPG strips frames and electrodes 5 Keepingthe right side of the frame slightly elevated place the left side of the frame against the mechanical stop about 2 cm or 1 inch from t
14. p 12 cm strip 24 well frame 12 well frame Protein OFFGEL Stock Solution 1 25X 0 96 mL 0 56 mL dH70 0 24 mL 0 14 mL Total Solution 1 2 mL 0 7 mL If you are using a multichannel pipettor you will want to make extra IPG Strip Rehydration Solution so there is excess in the reservoir tray 3 Prepare the OFFGEL Protein Sample Add the OFFGEL Stock Solution 1 25X to each sample in the amount indicated in Table 3 Table3 OFFGEL Protein Sample 24 cm strip 12 cm strip 24 well frame 12 well frame Protein OFFGEL Stock Solution 1 25X 2 88 mL 1 44 mL sample 0 72 mL 0 36 mL The final salt concentration in the diluted sample should not exceed 10 mM 6 OFFGEL Fractionator Kit User Guide OFFGEL Fractionator Kit User Guide Procedures To prepare solutions and sample for Protein Fractionation Multiple Runs If you will be doing more than one run at a time or if you are running the same sample in multiple lanes you can use Table 4 and Table 5 to make multiple batches of the rehydration buffer and sample Table4 24cm 24 wells Stock Solution 1 25X 0 96mL 192mL 3 84mL 7 68 mL dH70 0 24mL 048mL 096mL 192mL Stock Solution 1 25X 2 88mL 5 76mL 11 52mL 23 04mL Sample 0 72mL 144mL 2 88mL 5 7 6mL Table5 12 cm 12 wells Stock Solution 1 25X 0 56mL 1 12mL 2 24mL 448mL dH70 0 14mL 0 28mL 0 56mL 1 12mL Stock Solution 1 25X 144mL 2 88mL 576mL 11 52mL Sample 0 36mL 0 72mL 144mL 2 88mL
15. resis but may interfere with downstream analysis by mass spectrometry and are therefore not recommended For use with the 24 cm strip 24 well frame OFFGEL kit If your sample is dried prepare the required amount of 1X OFFGEL solution 2 88 mL 1 25X OFFGEL Stock Solution 0 72 mL dHy0 and resuspend your sample directly in this solution If your sample is in an OFFGEL compatible solution of less than 0 72 mL add 0 72 mL minus sample volume in mL mL dH50 to 2 88 mL 1 25X OFFGEL Stock Solution mix and then add your sample If your sample is in greater than 0 72 mL of an OFFGEL compatible solution concentrate the sample down to less than or equal to 0 72 mL Before you begin e For use with the 12 cm strip 12 well frame OFFGEL kit If your sample is dried prepare the required amount of 1X OFFGEL solution 1 44 mL 1 25X OFFGEL Stock Solution 0 36 mL dHy0 and resuspend your sample directly in this solution If your sample is in an OFFGEL compatible solution of less than 0 36 mL add 0 36 mL minus sample volume in mL mL dH50 to 1 44 mL 1 25X OFFGEL Stock Solution mix and then add your sample If your sample is in greater than 0 36 mL of an OFFGEL compatible solution concentrate the sample down to less than or equal to 0 36 mL e Proteins in the sample need to be fully denatured reduced and solubilized prior to fractionation The Protein OFFGEL Solution contains Urea DTT and Thiourea to accomplish this 4
16. rument and reagent kits are functioning correctly the beta lactoglobulin should be in the following OFFGEL fractions 12 cm pH 3 10 IPG strip well 3 12 cm pH 4 7 IPG strip well 4 24 cm pH 3 10 IPG strip well 5 6 24 cm pH 4 7 IPG strip well 7 8 9 See Figure 1 on page 21 20 OFFGEL Fractionator Kit User Guide Procedures To run a test sample kDa kDa Ladder total load Al AZ 43 At AS 46 A 48 A9 m a a a a it 9r ST 55 63 63 46 m 46 28 28 15 es 15 6 5 Mm 6 5 ce ee Ree rr lb eF gt Fcfrc 35 16 m i i a n i o U a 1 6 L 1 2 3 4 5 6 7 8 9 10 Figure 1 Beta lactoglobulin Protein Test Sample in well 3 of a 12 cm pH 3 10 IPG strip Gel like image from an Agilent 2100 BioAnalyzer analysis of wells 1 through 9 using the Protein 80 Chip Kit 6 uL of each OFFGEL fraction were used per analysis without additional sample preparation Lane 1 shows the di luted sample prior to fractionation Lanes 2 through 10 show OFFGEL fractions 1 through 9 OFFGEL Fractionator Kit User Guide 21 Reference Reference Product Information Part Number Product Description 5188 6424 5188 6425 5188 6426 5188 6427 5188 6444 5188 6428 5188 8012 5188 8013 G3100 60007 G3100 60002 G4208 68700 3100 OFFGEL High Res Kit pH 3 10 3100 OFFGEL Low Res Kit pH 3 10 3100 OFFGEL High Res Kit pH 4 7 3100 OFFGEL Low Res
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