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User Guide for Reversed

1. 3 micro bed format pi pette tips dispense 0 5 to 2 pL of elution solution into a clean vial CAUTION Acetonitrile and methanol are volatile and evaporation can occur rapidly If this occurs add more eluant to recover sample Carefully aspirate and dispense eluant through ZipTip pipette tip at least three times without in troducing air into the sample Sample recovery can be improved at the expense of concentra tion by increasing elution volume to 5 pL For Direct Spotting onto a MALDI TOF MS Target Elute with or without matrix in elution solution 1 Pipette 0 5 to 4 pL of desalted concentrated sample directly onto target by depressing plunger until appropriate volume is dispensed 2 Save or discard the remaining sample ZipTip Pipette Tips User Guide 7 For Nanoelectrospray MS Sample can be eluted into clean vial or using a GELoader tip Eppendorf cat no 0030 001 222 into a nanospray needle 1 Cut the GELoader tip about 2 3 mm above where the tip is fused to its capillary end GELoader tip Collen UD 2 Before the final dispense firmly press the cut down GELoader tip onto the ZipTip pipette tip with a slight twisting motion gt lt ZipTip pipette tip GELoader tip D a The leak free fit allows elution directly into a nanospray needle 8 www millipore com ziptip For Fractionating Peptides or Proteins 1 Pipette 1 to 3
2. Alcohol 100 Mercaptoethanol 0 1 M Mercaptoethanol 1 0 M Methyl Alcohol 100 Methyl Ethyl Ketone 100 Methyl Isobutyl Ketone 100 Nitric Acid 0 1 N Nitric Acid 1 5 N Phenol 0 5 Phosphoric Acid 1M Sodium Azide 1 Sodium Hydroxide 0 5 N Sodium Hydroxide 0 1 N Sodium Hypochlorite 100 ppm Sodium Hypochlorite 200 ppm Sulfuric Acid 1 Toluene 1 Triton X 100 Tween Urea 6 M VSS KSLSSSSLKSSLKRYSSLS ZipTip Pipette Tips User Guide 11 Troubleshooting Sample preparation problems with ZipTip stan dard and micro bed formats and ZipTip pipette tips can be divided into two categories m sample does not bind m sample binds but is not recovered The following table outlines common problems and their possible causes and suggests procedures to solve those problems The procedures may or may not work with your ZipTip application Incomplete binding is the problem most often en countered when performing sample preparation To determine whether the problem is binding or elution the best approach is to try the first three procedures for incomplete binding in the follow ing table Then use a step gradient approach to elution First elute the tip with 50 ACN in 0 1 TFA Then repeat the elution on the same tip with 75 ACN in 0 1 TFA This method addresses both types of problems using a single sample If the problems persist contact Millipore Techni cal Service fo
3. pL of 0 1 TFA 5 acetonitrile into clean vial Perform 3 4 aspirate dispense cycles to elute hydrophilic peptides or pro teins from tip Use the final dispense cycle to apply peptides or proteins directly to target Wash tip immediately by aspirating 0 1 TFA 5 acetonitrile Dispense to waste Repeat twice Perform next step gradient e g 5 10 20 30 or 50 ACN by increasing acetonitrile and repeat steps 1 and 2 until step gradient is com pleted NOTE Thoroughly wash the tip with respec tive eluant prior to increasing ACN elution to minimize peptide or pro tein carry over ZipTip Pipette Tips User Guide 9 Chemical Compatibility v Acceptable Long exposures at room temperature have no significant effect Questionable Short exposures at room temperature cause little or no damage X Not recommended Short exposure may cause permanent damage Reagent ZipTip Pipette Tips Acetic Acid Glacial Acetone Acetonitrile 100 Aliphatic Esters Ammonium Hydroxide 5 Benzene 100 n Butanol 100 Butyl Acetate Chloroform 1 Dichlorobenzene 100 Dichloromethane 1 Diethanolamine 5 Dimethyl Acetamide 100 Dimethyl Formamide 100 Dimethyl Formamide 1 Ethanol 100 Formic Acid 5 Guanidine HCl 6 M Hydrochloric Acid CSS NN KK YK SKS KRY RK 10 www millipore com ziptip Chemical Compatibility continued Reagent ZipTip Pipette Tips Hydrogen Peroxide Isopropyl
4. User Guide for Reversed Phase Ziplip Pipette Tips for Sample Preparation MILLIPORE Notice The information in this document is subject to change without notice and should not be construed as a commitment by Millipore Corporation Millipore Corporation assumes no re sponsibility for any errors that may appear in this document This manual is believed to be complete and accurate at the time of publication In no event shall Millipore Corporation be liable for incidental or consequential damages in connection with or arising from the use of this manual P36110 Rev G 05 05 2005 Millipore Corporation All rights reserved Millipore is a registered trademark of Millipore Corporation ZipTip and Milli Q are registered trademarks of Millipore Corpo ration GELoader is a trademark of Eppendorf Netheler Hinz GmbH Tween is a registered trademark of ICI Americas Inc Triton is a registered trademark of Union Carbide Corporation Boihit Proline is a trademark of Biohit Oy Introduction The ZipTip pipette tip is a 10 pL pipette tip with a bed of chromatography media fixed at its end It is intended for concentrating and purifying pep tide protein or oligonucleotide samples These instructions describe the use of ZipTip pi pette tips containing C and C reversed phase media for desalting and concentrating peptides and proteins For information on concentrating and desalt ing oligonucleotide samples request Millipo
5. ion solution to a maximum of 75 90 ACN v v in 0 1 TFA Sample not freely soluble Decrease ACN concentration to in ACN 20 40 in a 0 1 TFA or suitable ion pairing agent 14 www millipore com ziptip Specifications Materials of Construction Pipette tip Polypropylene Media Cis spherical silica 15 pm 200A pore size C spherical silica 15 pm 300A pore size Tip volume 10 pL Adsorptive bed C standard bed format 0 6 pL C micro bed format 0 2 uL C 0 6 pL Length 31 mm 1 22 in Capacity when used with saturating amounts of analyte C Standard bed format 2 1 0 pg typically 5 0 pg C micro bed format typically 2 0 pg C 2 0 5 pg typically 3 3 pg Max Temperature 70 C Min Temperature 4 C Working pH Range 2 to 13 ZipTip Pipette Tips User Guide 15 Ordering Information ZipTip Resin Catalogue Pack Type Number 8 pack C standard bed ZTC 18S 008 resealable C Gmicro bed ZTC 18M 008 C ZTC 048 008 24 pack C standard bed ZTC 18S 024 resealable C micro bed ZTC 18M 024 C ZTC 04S 024 96 pack C standard bed ZTC 18S 096 tip rack C micro bed ZTC 18M 096 C ZTC 04S 096 960 pack C standard bed ZTC 18S 960 10 x 96 C micro bed ZTC 18M 960 tip rack C ZTC 04S 960 For a complete listing of available ZipTip chemis tries visit www millipore com ziptip or contact your local Millipore office 16 www millipore com ziptip Technical As
6. iring agents Ensure that the final sample solution has a pH lt 4 Optimal binding of protein to the ZipTip pipette tip may also require a chaotropic agent e g guanidine HCl at a final concentration of 1 6M If the sample does not already contain chaotropic salts add them a few minutes before binding These salts will be removed during the wash step following sample binding ZipTip Pipette Tips User Guide 5 Equilibrate the ZipTip Pipette Tip for Sample rage Depress pipettor plunger to a dead stop Us ing the maximum volume setting of 10 pL aspirate wetting solution into the tip Dis pense to waste Repeat Aspirate equilibration solution Dispense to waste Repeat Bind and Wash the Peptides or Proteins Follow these steps after equilibrating the ZipTip pipette tip 1 Bind peptides and or proteins to ZipTip pi pette tip by fully depressing the pipette plunger to a dead stop Aspirate and dispense the sample 7 10 cycles for maximum binding of complex mixtures Aspirate wash solution into tip and dispense to waste Repeat at least once NOTE A 5 methanol in 0 1 TFA water wash can improve desalting effi ciency Additional washing may be required for electrospray MS 6 www millipore com ziptip Elute the Peptides or Proteins For ZipTip standard bed format and ZipTip pipette tips dispense 1 to 4 pL of elution solu tion into a clean vial using a standard pipette tip In the case of ZipTip
7. mptly of any such breach If after exercising reasonable efforts Millipore is unable to repair or replace the product or part then Millipore shall refund to the customer all monies paid for such applicable product or part MILLIPORE SHALL NOT BE LIABLE FOR CONSEQUENTIAL INCIDENTAL SPECIAL OR ANY OTHER INDIRECT DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY CUSTOMER FROM THE USE OF ITS PRODUCTS MILLIPORE
8. r further suggestions 12 www millipore com ziptip Incomplete Binding Possible Cause C C beads dewetted before sample was applied The hydrophobic beads can de wet in less than a minute Suggested Procedure After wetting with ACN flush the tip with 0 1 TFA and leave the plug immersed in liquid until immediately before sample binding Sample was not sufficiently acidified with TFA The pH should be below 4 The TFA concentration should be between 0 1 1 0 Spike sample with a few microliters of 0 5 1 TFA Sample not freely soluble Add Guanidine HCl to the sample to achieve a final concentration between 1 6M Guanidine actually enhances binding by helping to wet the hydrophobic surface and reducing polypeptide secondary structure Sample too hydrophilic for adsorption Relatively few options for solving this problem The best approach is to use a ZipTip pipette tip instead of a reversed phase tip Sample amount too low for detection Make sure samples are within the detection limits of the instrument In general a good MS signal should be obtained with 1 picomole of sample e g 5 pL of a 0 2 picomole pl solution Substantially lower amounts can be detected if the sample is clean ZipTip Pipette Tips User Guide 13 Incomplete Elution Possible Cause Suggested Procedure Sample tenaciously adsorbed Increase acetonitrile content of to the C C particles desorpt
9. re publication TN225 NOTE Because the adsorptive bed provides a slight back pressure do not use the ZipTip pi pette tip for accurate volumetric dispensing To achieve optimal sample uptake and de livery set the pipettor to 10 pL and attach the ZipTip pipette tip securely Throughout the procedure depress and release the plunger slowly to ensure optimal movement of solution through the resin bed 2 www millipore com ziptip Materials The following table outlines the solutions required for use with ZipTip pipette tips containing C and C media C is offered in two bed volumes m ZipTip tips a standard bed of 0 6 pL for sample elution in 1 to 4 pL m ZipTip cs tips a micro bed of 0 2 pL for elution in lt 1pL The procedure also requires a compatible 10 pL pipettor For simultaneous processing of multiple samples Millipore recommends the Biohit Proline Multi channel Pipettor Solution ZipTipcis ciePipette Tips ZipTip Pipette Tips Wetting solution 100 acetonitrile ACN 100 acetonitrile ACN Sample Adjust sample to 0 1 Adjust sample to 0 1 preparation trifluoroacetic acid TFA trifluoroacetic acid TFA final sample pH should final sample pH should be lt 4 be lt 4 Optional Guanidine HCI 1 6M may be added Equilibration 0 1 TFA in Milli Q 0 1 TFA in Milli Q solution grade water grade water Wash solution 0 1 TFA in Milli Q 0 1 TFA in Milli Q grade water grade
10. sistance For more information contact the Millipore office nearest you In the U S call 1 800 MILLIPORE 1 800 645 5476 Outside the U S see your Millipore catalogue for the phone number of the office nearest you or go to our web site at www millipore com offices for up to date worldwide contact information You can also visit the tech ser vice page on our web site at www millipore com techservice ZipTip Pipette Tips User Guide 17 Standard Warranty Millipore Corporation Millipore warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products MILLIPORE MAKES NO OTHER WAR RANTY EXPRESSED OR IMPLIED THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PUR POSE The warranty provided herein and the data specifications and descriptions of Millipore products appearing in Millipore s published catalogues and product literature may not be altered except by express written agreement signed by an officer of Mil lipore Representations oral or written which are inconsistent with this warranty or such publications are not authorized and if given should not be relied upon In the event of a breach of the foregoing warranty Millipore s sole obligation shall be to repair or replace at its option the applicable product or part thereof provided the customer noti fies Millipore pro
11. water Elution solution 0 1 TFA 50 ACN 0 1 TFA 50 75 ACN with or without matrix with or without matrix For electrospray elute with 1 formic acid 50 methanol For fractionating peptides prepare varying concentrations of ACN water e g 5 10 36 30 End 50 with or without 0 1 TFA ZipTip Pipette Tips User Guide 3 Guidelines for Selecting ZipTip or ZipTip Pipette Tips ZipTipcie pipette tip ZipTipca pipette tip o 50 000 100 000 Peptide Protein Molecular Weight most applicable applicable but not ideal E not recommended ZipTip pipette tips are most applicable for pep ides and low molecular weight proteins while ZipTip pipette tips are most suitable for low to intermediate molecular weight proteins In many cases the two devices can be used interchange ably Because higher molecular weight proteins tend to adsorb tenaciously to hydrophobic sur faces ZipTip pipette tips are recommended for proteins over 100 000 MW 4 www millipore com ziptip Procedures for Use The following procedures describe how to pre pare the sample and equilibrate the ZipTip pipette tips for sample binding washing and elution See the Materials section for information on the ap propriate solutions for your application Prepare the Sample Maximum binding to the ZipTip pipette tip is achieved in the presence of TFA 0 1 or other ion pa

User Guide for Reversed

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