TargetSeq™ Exome Enrichment System User Guide
Contents
1. Product Information iBind Flex Western Device System components Contents Components The iBind Flex Western System is a benchtop device utilizing sequential lateral flow SLF to perform hands free blocking antibody binding and washes for western detection workflows The iBind Flex Western System uses no external power source and relies on mechanical pressure from the iBind Flex Western Device on a iBind Flex Card to generate the sequential flow of immunodetection reagents for performing the blocking antibody binding and wash steps involved in western detection workflows The iBind Flex Western System consists of e iBind Flex Western Device e iBind Flex Midi Insert e iBind Flex Mini Insert e iBind Flex Multi Strip Insert e iBind Flex Cards e iBind Flex Solution Kit e iBind Flex Fluorescent Detection FD Solution Kit The components included with the iBind Flex Western Device Cat no SLF2000 are listed below Bind Flex Western Device iBind Blotting Roller Bind Flex Midi Insert Bind Flex Mini Insert Bind Flex Multi Strip Insert IBind Flex Western Device User Guide Required materials The following components are used with the iBind Flex Western System but not supplied with not included with the iBind Flex Western Device the device iBind Flex Cards The iBind Flex Card is a unique matrix optimized for homogenous fl2. e g use 1 1000 dilution if 1 5000 dilution is recommended 12 IBind Flex Western Device User Guide Fluorescent Detection Procedure Experimental Use the following protocol when using the iBind Flex Western System in overview conjunction with the LI COR Odyssey Imaging System Perform antibody binding using i e Midi Insert e Mint Insert e Multi Strip Insert Prepare 1X iBind 1X iBind FD Flex Solution is used for blocking diluting antibodies washing Flex FD Solution and wetting the iBind Flex Card e The Standard 1X iBind Flex FD Solution is recommended for use with most primary antibodies e Use the Optional 1X iBind Flex FD Solution only if initial results give low sensitivity or high background Prepare 50 mL of 1X iBind Flex FD Solution for each run as follows Reagent Volume Standard Optional iBind Flex FD 5X Buffer 10 mL 2 9 ML iBind Flex 100X Additive 125 uL 900 uL Distilled Water 39 9 mL 47 mL Total 90 mL 90 mL Prepare Store membranes in 1X iBind Flex FD Solution in distilled water or dry if membranels they are not used immediately after transfer Membranes should only be blocked with 1X iBind Flex FD Solution Before performing the antibody binding prepare the membrane s as follows e Pre activate PVDF membranes in 100 methanol and rinse in distilled water e Immerse the blotted membrane protein side up in 1X iBind Flex FD Solution Use 10 mL for ea
3. Mini Blot Vertically Cut Strip s 1X iBind Flex iBind Flex FD Solution Diluted secondary antibody 1X iBind Flex iBind Flex FD Solution If not all lanes are being used to process vertically cut strips add the volume of water given for each row to the wells in the unused lane s Mini Insert Multi Strip Insert Close the Well Cover and incubate 2 5 hours or longer Note The membrane can be left in the iBind Flex Western Device overnight but perform detection as soon as possible to avoid loss of sensitivity Open the Well Cover to verify that the well s in row 4 are completely empty indicating that the run is over before releasing the latch handle and opening the lid After incubation rinse the membrane twice in 20 mL of distilled water for 2 minutes and proceed to the appropriate detection protocol page 24 25 IBind Flex Western Device User Guide 37 38 IBind Flex Western Device User Guide
4. X ray film OR Scan the membrane sandwich in a digital imager IBind Flex Western Device User Guide Chemiluminescent detection with horse radish peroxidase Fluorescent detection The following protocol describes performing chemiluminescent detection on a membrane using the Novex ECL Chemiluminescent Substrate Reagent Kit after blocking antibody binding and washes have been completed by the iBind Flex Western Device Light emission is most intense from 5 30 minutes after membrane incubation and decreases slowly with time over the course of several hours Prepare chemiluminescent substrate solution Scale volumes according to the amount needed for the type of membrane being processed HRP Chemiluminescent Substrate 1 25 mL Reagent A HRP Chemiluminescent Substrate 1 25 mL Reagent B Place the membrane on a sheet of transparency plastic with the protein side up Do not allow the membrane to dry out With a clean pipette apply the appropriate volume of chemiluminescent substrate solution evenly across the membrane surface do not touch the membrane surface with the pipette Incubate for 1 minute Blot excess chemiluminescent substrate solution from the membrane surface with filter paper Do not allow the membrane to dry out Cover the membrane with another clean piece of transparency plastic or plastic wrap Place a piece of X ray film over the membrane sandwich and expose for 1 second to sever
5. 20 23 28 IBind Flex Western Device User Guide Blot improperly placed on iBind e Place the membrane in the designated Flex Card Membrane Region on the iBind Flex Card High background continued e The protein side of the blot should be in contact with the iBind Flex Card e The low MW regions should be closest to the Stack e The membrane should not be in contact with the Stack Card stack wet prior to run Ensure that 10 mL of 1X iBind Flex iBind Flex FD Solution is added to the flow region of the card Avoid adding the solution to the Stack Improper preparation of iBind Prepare 1X iBind Flex iBind Flex FD Flex iBind Flex FD Solution Solutions as directed pages 12 13 Improper application of solutions Add the appropriate solutions for each well in to iBind Flex Wells numerical order pages 17 20 23 Blot improperly placed on iBind e Place the membrane in the designated Flex Card Membrane Region on the iBind Flex Card e The protein side of the blot should be in contact with the iBind Flex Card e The low MW regions should be closest to the Stack e The membrane should not be in contact with the Stack Card stack wet prior to run Ensure that 10 mL of 1X iBind Flex iBind Flex FD Solution is added to the flow region of the card Avoid adding the solution to the Stack Membrane contaminated by Wear clean gloves at all times and use forceps fingerprin
6. 4 are completely empty indicating that the run is over before releasing the latch handle and opening the lid After incubation rinse the membrane twice in 20 mL of distilled water for 2 minutes and proceed to the appropriate detection protocol page 24 25 Discard iBind Flex Card after use IBind Flex Western Device User Guide Using the iBind Flex Western Device with the Multi Strip Insert Prepare iBind Open the lid of the iBind Flex Western Device 1 Flex Card 2 Verify the Multi Strip Insert is inserted in the iBind Flex Western Device 3 Open the packaging for the iBind Flex Card 4 Hold the iBind Flex Card by the Stack and remove the card from the packaging 5 Place the iBind Flex Card on the Stage 6 Pipette 10 mL of 1X iBind Flex iBind Flex FD Solution evenly across the Flow Region Multi Strip Insert 7 Pipette 2 mL of 1X iBind Flex iBind Flex FD Solution so that it pools along the center of the membrane region on the iBind Flex Card IBind Flex Western Device User Guide 21 Place membrane 1 Place the membrane on top of the pooled solution with the protein side on iBind Flex down and the low molecular weight protein region closest to the Stack Card Note the membrane should not come in contact with the stack Membranes in all lanes Membranes in some lanes o ee E E ea 2 Use the blotting roller to remove any air bubbles between the membra
7. FD Solution so that it pools at the center of the membrane region on the iBind Flex Card Do 4 an lt Go l n N y gt P b a ty A ys y i lt a i Y TOA gi Ge ae bo Farge ay iw 3 er A a Ra 7 O mr a A ie e aA PR 22 PE J 4 2s A 5 IBind Flex Western Device User Guide 15 Place membrane 1 Place the membrane on top of the pooled solution with the protein side on iBind Flex down and the low molecular weight protein region closest to the Stack Card Note the membrane should not come in contact with the stack 2 Use the blotting roller to remove any air bubbles between the membrane and the iBind Flex Card Midi Insert 3 Make sure that the the membrane is within the boundaries of the membrane region No part of the membrane should be directly under the Midi Insert 4 Lower the lid of the iBind Flex Western Device and close the latch handle to lock the lid 16 IBind Flex Western Device User Guide Add solutions to wells 1 Open the Well Cover and add solutions sequentially to the iBind Flex Wells starting with row 1 Diluted secondary antibody 1X iBind Flex iBind Flex FD Solution Close the Well Cover and write the start time of incubation on the lid of the iBind Flex Western device Incubate 2 5 hours or longer Note The membrane can be left in the iBind Flex Western Device overnight but perfo
8. OC COUN C a6 i rcnaiarets ern rE EEEE EEE ETSE EES 11 Fluorescent Detection Procedure neueren i e E ET E E ETE E ETTE EN 13 Using the iBind Flex Western Device with the Midi Insert 00 0 0 cccccssseesssnneeeeeeeeeeeeeeeessssesssssssneeeeeeeeeees 15 Using the iBind Flex Western Device with the Mini Insert c ccssessssssssssssssesssseseseseseseeeeeeeseeeeeeees 18 Using the iBind Flex Western Device with the Multi Strip Insert cccccsssssssssssssssesssessseseeeeseeeeees 21 SRO CUO oes anti eis se ite a Pe ica hs ait De tig S 24 OD Ze COIN cerre E 26 IU SUIT PNG Covent acta Skt ta et wee ace ae oat a Oe te ole ae ttt 27 MET UH S DOGON ranne a Gee geen ems cara aap cement Sep nneeee gunk E enna adanhd emnneecees 28 ADDENDA inia E beiase a et ung eta awed Suete nna awe sts wedeinan E sew eSuceedeeseseeucarteees 32 Pe LAUe Gi OOUCUS eera aen icae hates lode seca meiane obese A E E E EA 32 TSEC AL SU O28 aaa ce dseneans deren aesentatgadeivnanatdatestatadataqaeseaustionsdemnnnateatebanndavaasaddstetatadatansesesntoosdamacatemusndanass 33 APPENA B aiia eee es es bee a at eeu pectic a ewan e dada wad ene Saeed andes a 34 PROGUG tO CICA WlOl Seana aia ita EEE a ee ETEEN AAE EE EO cee meine anaes A 34 ADDEN C oios a a a SE aa E a S A a uticea ele 35 Using the iBind Flex Western Device with an iBind Card cccccceeseseeessnnneeeeeeeeeeeeeeesesssssssnnnneeeeeeeeeees 39 IBind Flex Western Device User Guide 3
9. al minutes then develop the X ray film OR Scan the membrane sandwich in a digital imager The following protocol describes performing fluorescent detection on a membrane after blocking antibody binding and washes have been completed by the iBind Flex Western Device Rinse the membrane with water after completion of the run 2 Scan the membrane wet or dry with the LI COR Odyssey CLx imager using the appropriate channel on Auto resolution IBind Flex Western Device User Guide 25 Optimization Chemiluminescent After performing an initial experiment conditions can be optimized by varying detection the dilution of primary and secondary antibodies according to the following table Condition Observation Primary and Secondary Antibody Concentrations HRP Protocol strong signal Condition Observation Secondary Antibody Concentration Novex AP Chemiluminescent Detection Kit 1 1000 to 1 500 1 500 to 1 250 ani neae gt 1 4000 gt 1 2000 strong signal Start with a 1X concentration of primary antibody Further optimization by increasing the primary antibody concentration may be necessary depending upon your results Start with a 5X concentration of secondary antibody Further optimization by decreasing the secondary antibody concentration may be necessary depending upon your results Fluorescent The Standard 1X iBind Flex FD Solution is recommended for use with most detection primary antibodies and shou
10. as set forth in the Life Technologies General Terms and Conditions of Sale found on the Life Technologies web site at http www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies 33 Appendix B Product Specifications iBind Flex Western Device specifications iBind Flex Card specifications 34 Dimensions Material Operating Temperature Temperature Limit 30 0 cm I x 25 2 cm w x 8 0 cm h Aluminum plastic PC ABS steel silicone neodymium magnets 18 C to 30 C 30 C The iBind Flex Western Device is impervious to alcohol but not compatible with chlorinated hydrocarbons e g chloroform aromatic hydrocarbons e g toluene benzene or acetone Dimensions Material Operating Temperature Temperature Limit 17 8 cm 1 x 17 8 cm w x 0 8 cm stack height Glass fiber 18 C to 30 C 30 C IBind Flex Western Device User Guide Appendix C Using the iBind Flex Western Device with an iBind Card Prepare iBind 1 Open the lid of the iBind Flex Western Device Card 2 Verify the Mini Insert or Multi Strip Insert is inserted in the iBind Flex Western Device depending upon the type of blot being processed 3 Open the packaging for the iBind Card Hold the iBind Card by the Stack and remove the card from the packaging 5 Place the iBind Card on the Stage in the position corresponding to lan
11. ch mini sized membrane or for vertically cut strips or 20 mL for midi sized membranes IBind Flex Western Device User Guide 13 Prepare primary Dilute antibodies with 1X iBind Flex FD Solution antibody solution 1 Antibody Dilute antibody to manufacturer s recommended dilution Prepare secondary _ Dilute antibodies with 1X iBind Flex FD Solution antibody solution SDS is added to a final concentration of 0 05 to reduce background signal particularly when using PVDF membranes or IRDye 680LT secondary antibodies iBind Flex FD 10 SDS 20 uL 10 uL 3 5 uL Alexa Fluor 680 OR 2 uL 1 2000 1 uL 1 2000 0 35 uL 1 2000 IRDye 680LT 1 uL 1 4000 0 5 uL 1 4000 0 18 uL 1 4000 Alexa Fluor 790 OR 2 uL 1 2000 1 uL 1 2000 0 35 uL 1 2000 IRDye 800CW 1 3 uL 1 3000 0 67 uL 1 3000 0 23 uL 1 3000 14 IBind Flex Western Device User Guide Using the iBind Flex Western Device with the Midi Insert Prepare iBind 1 Open the lid of the iBind Flex Western Device Flex Card 2 Verify the Midi Insert is inserted in the iBind Flex Western Device 3 Open the packaging for the iBind Flex Card 4 Hold the iBind Flex Card by the Stack and remove the card from the packaging 5 Place the iBind Flex Card on the Stage 6 Pipette 10 mL of 1X iBind Flex iBind Flex FD Solution evenly across the Flow Region Midi Insert 7 Pipette 2 mL of 1X iBind Flex iBind Flex
12. ct with the iBind Flex Card immediately after adding a 10 mL pool of 1X iBind Flex iBind Flex FD Solution Use the roller provided to ensure proper contact 30 IBind Flex Western Device User Guide Weak or no Device opened prior to The device should not be opened once the card signal continued completion of run has been placed in the device Re sealing of the wells on the card can result in leaks Sample improperly prepared SDS and reducing agents may interfere with antigenicity compromised some antibody antigen affinities Sample too dilute Load a higher concentration or amount of protein onto the gel Protein weakly bound to Ensure that transfer buffer contains 10 20 membrane methanol Insufficient exposure time Re expose film for a longer period of time Insufficient substrate incubation Perform each step for the specified amount of time or remove blot from substrate when signal to noise ratio is acceptable Substrate is contaminated Wear gloves at all times and keep bottles tightly capped when not In use Blots are too old Protein may have broken down over time Use freshly prepared blots Large scattered Protein is overloaded Reduce load or dilute concentration of sample agian Poor or incomplete transfer Refer to Western Blotting instructions IM 9051 and repeat blot Primary antibody is too Follow the supplier s recommended dilution or concentrated determine the optimum co
13. e Remove installan The iBind Flex well inserts are designed so that they only fit into the lid of the iBind Flex well iBind Flex Western Device in the correct orientation To install an insert Open the lid and well cover of the Open the lid and well cover of the iBind Flex Western Device iBind Flex Western Device 2 Push the insert from the back of 2 Slide the insert into the slot in the the lid and slide it out of the slot lid from the underside of the lid Blotting Roller The Blotting Roller is a plastic roller attached to a stainless steel handle 8 6 cm wide The Blotting Roller is used to remove any air bubbles between the membrane and the iBind Flex Card iy amp 4 eg os Pay A IBind Flex Western Device User Guide 7 iBind Flex Card The iBind Flex Card is a unique glass fiber matrix optimized for homogenous flow of immunodetection reagents The card consists of a Flow Region and a Stack Solutions in the well inserts are released from the wells and wicked towards the Stack via SLF IMPORTANT e Do not bend the iBind Flex Card e iBind Flex Cards are single use only Discard card after use The iBind Flex Card is placed on the iBind Flex Western Device so that it fits between the alignment tabs with the stack facing the front of the device Alignment Tabs by Thermo Fisher Scientific iBind Flex Card Invitrogen When the flow region is wet with so
14. e 1 of the Mini Insert or lanes 1 3 of the Multi Strip Insert 6 Pipette 5 mL of 1X iBind Flex iBind Flex FD Solution evenly across the Flow Region 7 Pipette 1 mL of 1X iBind Flex iBind Flex FD Solution so that it pools at the center of the membrane region on the iBind Card w r dk We eR Ji n i J a a d Pog b bA a i a IBind Flex Western Device User Guide 35 Place membrane 1 Place the membrane s on top of the pooled solution with the protein side on iBind Card down and the low molecular weight protein region closest to the Stack Note the membrane should not come in contact with the stack Mini size membrane Vertically cut strips Mini Insert 2 Use the blotting roller to remove any air bubbles between the membrane and the iBind Card Mini size membrane Vertically cut strips Mini Insert ve Multi Strip Insert pa Pze n Preys r Se D Oi 3 Make sure that the iBind Card is flush against the alignment tabs and that the membrane is within the boundaries of the membrane region No part of the membrane should be directly under the well insert 4 Lower the lid of the iBind Flex Western Device and close the latch handle to lock the lid 36 IBind Flex Western Device User Guide Add solutions to wells 1 Open the Well Cover and add solutions sequentially to the iBind Flex Wells starting with row 1 Solution Volume Well
15. ent for 10 midi blots or 20 mini blots iBind Elex 5X Buffer 100 mL Bno Flex 100X Additive ATT il IBind Flex Western Device User Guide 5 Description of Parts iBind Flex Western Device iBind Flex Western Device lid The iBind Flex Western Device is an automated device that utilizes sequential lateral flow SLF to automatcally perform blocking washing and antibody incubation steps in a western detection workflow SLF allows the timely release and flow of solutions and antibodies to the membrane without need of an external power source Each solution is released from iBind Flex wells to an iBind Flex Card via SLF The glass fiber matrix of the card allows for homogenous and consistent flow of the solutions to the membrane increasing the antigen antibody interaction The iBind Flex well inserts have four rows of wells for loading blocking solution antibodies and wash solutions There are three different well inserts for processing one midi blot up to two mini blots or up to six vertically cut strip blots Lid and Drawer Closed The lid of the iBind Flex Western Device is designed to be marked with standard lab markers A section is provided to mark the device as being in use and record the time at which an incubation is started iBind Flex IBind Flex Western Device User Guide The iBind Flex Western Device consists of a metallic stage made up of three sections The fron
16. hould not come in contact with the stack One mini membrane in lane 1 Two mini membranes in lanes 1 and 2 2 Use the blotting roller to remove any air bubbles between the membrane and the iBind Flex Card One mini membrane in lane 1 Two mini membranes in lanes 1 and 2 T EN gt Mini Insert Membrane Membrane a Region a gt Region 3 Make sure that the the membrane s are aligned with the lane s and within the boundaries of the membrane region No part of the membrane should be directly under the Mini Insert 4 Lower the lid of the iBind Flex Western Device and close the latch handle to lock the lid IBind Flex Western Device User Guide 19 Add solutions to wells 20 1 Open the Well Cover and add solutions sequentially to the iBind Flex Wells starting with row 1 Diluted primary antibody 1X iBind Flex iBind Flex FD Solution Diluted secondary antibody 1X iBind Flex iBind Flex FD Solution If only a single mini size membrane is being processed add the volume of water given for each row to the wells in the unused lane Close the Well Cover and write the start time of incubation on the lid of the iBind Flex Western device Incubate 2 5 hours or longer Note The membrane can be left in the iBind Flex Western Device overnight but perform detection as soon as possible to avoid loss of sensitivity Open the Well Cover to verify that the well s in row
17. iBind Flex Western System For western detection of proteins on PVDF or nitrocellulose membranes Catalog no SLF2000 Rev Date 13 April 2015 Publication Number MAN0010927 For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice DISCLAIMER TO THE EXTENT ALLOWED BY LAW LIFE TECHNOLOGIES AND OR ITS AFFILIATE S WILL NOT BE LIABLE FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT IMPORTANT LICENSING INFORMATION These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses TRADEMARKS 2015 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified 2 IBind Flex Western Device User Guide Contents FOLIC UNC IPI cl GHONM eect c hess Sea ae Ea ae a ge ee i Ee a 4 EC SEMED LOMHON Noah Shcencsazeate rhs sactanassagsacncnaacacadeanaaesinanaetadnanne se nana santana re nacaecaceanaaesenancetadnenne sae neseamaaceaeteneenan 6 NUON S isco abd ce Sc ere eet dase De ee a a a aa as eat dade erence eels 11 Procedural VEN VICWW orrs scat ae ste eatehance dae tented E cea ce saath ak beau dt ck nce ata ected deattanttdeaes 11 FIRE anG AP LCCC CMON R
18. ion dish which ts small enough to allow thorough coverage of membrane to prevent drying out Membrane is contaminated Use only clean new membranes Wear clean gloves at all times and use forceps when handling membranes Film overexposed or became wet Decrease exposure time or allow signal to during exposure further decay Prevent leakage by encasing membrane in transparency film and blotting excess substrate from edges before exposure Solutions or incubation tray is Use clean glassware and purified water to contaminated prepare solutions Replace or clean the tray thoroughly with a glassware cleaning detergent Rinse thoroughly with purified water Wear clean gloves at all times Concentrated primary antibody Follow the supplier s recommended dilution or used determine the optimum concentration by dot blotting Incorrect chemiluminescent Make sure CDP Star without enhancer Is used substrate used for PVDF Blot is overdeveloped Follow recommended developing time or remove blot from substrate when signal to noise ratio is acceptable Ink used to label membrane Any labeling of the membrane with ink should be limited to the low MW region of the blot Improper preparation of iBind Prepare 1X iBind Flex iBind Flex FD Flex iBind Flex FD Solution Solutions as directed pages 12 13 Improper application of solutions Add the appropriate solutions for each well in to iBind Flex Wells numerical order pages 17
19. ld be used for all initial experiments e If initial results give low sensitivity switch from the Standard 1X iBind Flex FD Solution to the Optional 1X iBind Flex FD Solution page13 e If low sensitivity persists increase the primary antibody concentration from 1X 5X as needed 26 IBind Flex Western Device User Guide Maintenance General guidelines Rinse the iBind Flex well inserts under running water after each use and allow the well inserts to dry before additional usage e Handle well inserts with care e Store unused well inserts in the drawer in the iBind Flex Western Device e To maximize the life of the springs in iBind Flex Western Device store the device with latch unlocked and the lid open as shown below gt IBind Flex Western Device User Guide 27 Troubleshooting iBind Flex Card damaged Replace with new card Ensure that rolling of the membrane on the card Is limited to the area labeled membrane Run Times in Excess of 3 hours Stack wet prior to run Ensure that 10 mL of 1X iBind Flex iBind Flex FD Solution is added to the flat region of the iBind Flex Card Avoid adding solution to the Stack Improper preparation of iBind Prepare 1X iBind Flex iBind Flex FD Flex iBind Flex FD Solution Solutions as directed pages 12 13 High background Membrane not completely wet Follow instructions for pre wetting the membrane Use an Incubat
20. lution lines appear on the iBind Flex Card to assist in alignment of mini sized or vertically cut strip membranes with wells iBind Flex Card invitrogen _ by Thermo Fisher Scientific j _ Pi IBind Flex Western Device User Guide Methods Procedural Overview General guidelines Wear the proper protective equipment gloves laboratory coat eye protection when performing experiments e Handle well inserts with care and keep them stored in the drawer of the iBind Flex Western Device when not in use e If you mark your membrane s with ink mark the membrane s near the low molecular weight region e Caution Exercise care when closing the lid of the iBind Flex Western Device to avoid catching fingers e Important No part of the membrane s should be directly under the wells when the lid is closed e Do not move the iBind Flex Western Device or open the lid until the well s in row 4 are completely empty 2 5 hours or longer Note The membrane s can be left in the iBind Flex Western Device overnight if desired e 1X iBind Flex Solution is used for HRP and AP detection while 1X iBind Flex FD Solution is used for fluorescent detection HRP and AP Detection Procedure Experimental Use the following protocol when using the iBind Flex Western System with overview HRP or AP detection protocols Prepare 1X iBind Flex Solution Prepare diluted antibody solutions Perfor
21. m antibody binding using 4 e Midi Insert e Mint Insert e Multi Strip Insert IBind Flex Western Device User Guide 11 Prepare 1X iBind 1X iBind Flex Solution is used for blocking diluting antibodies washing and wetting the iBind Flex Card Prepare 50 mL of 1X iBind Flex Solution for each run as follows Flex Solution Reagent Volume iBind Flex 5X Buffer 10 mL iBind Flex 100X Additive 900 uL Distilled Water 39 5 mL Total 50 mL Prepare membrane Store membranes in 1X iBind Flex Solution in distilled water or dry if they are not used immediately after transfer Membranes should only be blocked with 1X iBind Flex Solution Before performing the antibody binding prepare the membrane as follows e Pre activate PVDF membranes in 100 methanol and rinse in distilled water e Immerse the blotted membrane protein side up in 1X iBind Flex Solution Use 10 mL for each mini sized membrane or for vertically cut strips or 20 mL for midi sized membranes Prepare primary Dilute primary antibodies with 1X iBind Flex Solution according to the enzyme antibody solution system being used for detection 1 Antibody Dilute antibody to manufacturer s recommended dilution Prepare secondary _ Dilute primary antibodies with 1X iBind Flex Solution according to the enzyme antibody solution system being used for detection 2 Antibody Prepare antibody at 5X the manufacturer s recommended dilution
22. ncentration by dot blotting Spotted Poor or incomplete transfer Refer to Western Blotting instructions IM membrane 9051 and repeat blot Membrane pads are dirty or Soak with detergent and rinse thoroughly with contaminated purified water before use Replace pads when they become worn or discolored Follow instructions for pre wetting the membrane Membrane Is contaminated by Wear clean gloves at all times and use forceps fingerprints or keratin proteins when handling membranes Always handle membranes around the edges Uneven blocking The incubation dish must be small enough to allow thorough coverage of membrane Ink used to label membrane Any labeling of the membrane with ink should be limited to the low MW region of the blot iBind Flex Card damaged Replace with new card Ensure that rolling of the membrane on the card Is limited to membrane region Membrane is not in proper Place the membrane on the iBind Flex Card contact with the iBind Flex Card immediately after wetting the card with 10 mL of 1X iBind Flex iBind Flex FD Solution Use the roller provided to ensure proper contact IBind Flex Western Device User Guide 31 Appendix A Related Products Additional products Many of the components of the iBind Flex Western System as well as additional reagents that may be used for electrophoresis of proteins are available separately from Life Technologies Ordering information is
23. ne and the iBind Flex Card Membranes in all lanes Membranes in some lanes Multi Strip en al J Insert f Membrane _ Membrane Region Region 3 Make sure that the the membrane s are aligned with the lane s and within the boundaries of the membrane region No part of the membrane should be directly under the Multi Strip Insert 4 Lower the lid of the iBind Flex Western Device and close the latch handle to lock the lid 22 IBind Flex Western Device User Guide Add solutions to wells 1 Open the Well Cover and add solutions sequentially to the iBind Flex Wells starting with row 1 Diluted primary antibody 1X iBind Flex iBind Flex FD Solution Diluted secondary antibody 1X iBind Flex iBind Flex FD Solution If not all lanes are being used to process vertically cut strips add the volume of water given for each row to the wells in the unused lane s Close the Well Cover and write the start time of incubation on the lid of the iBind Flex Western device Incubate 2 5 hours or longer Note The membrane can be left in the iBind Flex Western Device overnight but perform detection as soon as possible to avoid loss of sensitivity Open the Well Cover to verify that the well s in row 4 are completely empty indicating that the run is over before releasing the latch handle and opening the lid After incubation rinse the membrane twice in 20 mL of distilled water for 2 mi
24. nutes and proceed to the appropriate detection protocol page 24 25 Discard iBind Flex Card after use IBind Flex Western Device User Guide 23 Detection Chemiluminescent detection with alkaline phosphatase 24 The following protocol describes performing chemiluminescent detection on a membrane using the Novex AP Chemiluminescent Detection Kit after blocking antibody binding and washes have been completed by the iBind Flex Western Device 1 Prepare chemiluminescent substrate solution based on the type of membrane being used Scale volumes according to the amount needed for the type of membrane being processed Novex AP Chemiluminescent Substrate CDP Star 2 379 mL Chemiluminescent Substrate 0 125 mL Enhancer Nitro Block II 2 Place the membrane on a sheet of transparency plastic with the protein side up Do not allow the membrane to dry out 3 With a clean pipette apply the appropriate volume of chemiluminescent substrate solution evenly across the membrane surface do not touch the membrane surface with the pipette 4 Incubate for 5 minutes 5 Blot excess chemiluminescent substrate solution from the membrane surface with filter paper Do not allow the membrane to dry out 6 Cover the membrane with another clean piece of transparency plastic or plastic wrap 7 Place a piece of X ray film over the membrane sandwich and expose for 1 second to several minutes then develop the
25. ow of immunodetection reagents along its length see page 10 for details The iBind Flex Cards are single use and sold separately see page 32 for ordering details The components included with the iBind Flex Cards Cat no SLF2010 are listed below iBind Flex The iBind Flex Fluorescent Detection FD Solution Kit is used for preparing Fluorescent blocking dilution and washing buffers for the iBind Flex western detection Detection FD protocol in conjunction with Alexa Fluor or IRDye conjugated secondary antibodies The iBind Flex Fluorescent Detection FD Solution Kit is sold SO UHOREAN separately see page 32 for ordering details The components included with the iBind Flex Fluorescent Detection FD Solution Kit Cat no SLF2019 are listed below and sufficient for 10 midi blots or 20 mini blots Bind Flex FD 5X Buffer 100 mL Bind Flex 100X Additive 3x 1 7 mL iBind Flex FD 10 SDS 200 pL iBind Flex The iBind Flex Solution Kit is used for preparing blocking dilution and Solution Kit washing buffers for the iBind Flex western detection protocol using chemiluminescent or chromogentic substrates and alkaline phosphatase AP or horseradish peroxidase HRP conjugated secondary antibodies The iBind Flex Solution Kit is sold separately see page 32 for ordering details The components included with the iBind Flex Solution Kit Cat no SLF2020 are listed below and suffici
26. provided below For details visit www lifetechnologies com or call Technical Support page 33 Bolt 4 12 Bis Tris Plus Gel 10 Well ee NuPAGE Novex 4 12 Bis Tris Gels 10 gels NP0321BOX 1 0 mm 10 well a a Ane E NuPAGE Novex 4 12 Bis Tris Gels 10 gels NP0335BOX 1 5 mm 10 well 32 IBind Flex Western Device User Guide Technical Support Obtaining support Safety Data Sheets SDS Certificate of Analysis Limited warranty IBind Flex Western Device User Guide For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Safety Data Sheets SDSs are available at www lifetechnologies com support The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available from www lifetechnologies com Life Technologies and or its affiliate s warrant their products
27. purified water when preparing reagents Protein of interest ran off the gel Match gel separation range to size of protein being transferred Poor retention of proteins Match gel separation range to size of protein being transferred Use a molecular weight marker with relevant size proteins Larger proteins require more transfer time smaller proteins less Use membrane with the appropriate binding capacity see Western Blotting instructions Improper preparation of iBind Prepare 1X iBind Flex iBind Flex FD Flex iBind Flex FD Solution Solutions as directed pages 12 13 Improper application of solutions Ensure that the solutions are added to the to iBind Flex Wells correct wells and that the wells are loaded In numerical order Blot improperly placed on iBind Ensure that the protein side of the blot Is in Flex Card contact with the iBind Flex Card and is placed in the region labeled membrane Stack wet prior to run Ensure that 10 mL of 1X iBind Flex iBind Flex FD Solution is added to the flat region of the iBind Card Avoid adding solution to the Stack Cross contamination of solutions Do not move the iBind Flex Western Device in wells during the run iBind Flex Card damaged Replace with new card Ensure that rolling of the membrane on the card Is limited to the area labeled membrane Membrane Is not in proper Place the membrane on the iBind Flex Card conta
28. rm detection as soon as possible to avoid loss of sensitivity Open the Well Cover to verify that row 4 is completely empty indicating that the run is over before releasing the latch handle and opening the lid After incubation rinse the membrane twice in 20 mL of distilled water for 2 minutes and proceed to the appropriate detection protocol page 24 25 Discard iBind Flex Card after use IBind Flex Western Device User Guide 17 Using the iBind Flex Western Device with the Mini Insert Prepare iBind Open the lid of the iBind Flex Western Device 1 Flex Card 2 Verify the Mini Insert is inserted in the iBind Flex Western Device 3 Open the packaging for the iBind Flex Card 4 Hold the iBind Flex Card by the Stack and remove the card from the packaging 5 Place the iBind Flex Card on the Stage 6 Pipette 10 mL of 1X iBind Flex iBind Flex FD Solution evenly across the Flow Region Mini Insert 7 Pipette 1 mL of 1X iBind Flex iBind Flex FD Solution in each lane to be used for a membrane so that it pools at the membrane region on the iBind Flex Card Membrane Region g re gt oe lt y R e 7 lt 18 IBind Flex Western Device User Guide Place membrane 1 Place the membrane on top of the pooled solution with the protein side on iBind Flex down and the low molecular weight protein region closest to the Stack Card Note the membrane s
29. t and rear sections of the stage are spring plates designed to apply specific amounts of pressure on an iBind Flex Card placed on the stage when the lid of the device is locked The pressure on the iBind Flex Card results in the sequential flow of immunodetection reagents from the wells in which they are loaded The flow rate is highly reproducible because the amount of pressure and the viscosity of the fluids remain constant Lid and Drawer Open Well Insert Latch Handle Drawer for storing The iBind Flex well inserts are stored in a drawer at the front of the device iBind Flex well Press on the front of the drawer to release the latch and slide the drawer open inserts IBind Flex Western Device User Guide iBind Flex Midi Insert iBind Flex Mini Insert iBind Flex Multi Strip Insert The iBind Flex Midi Insert is designed for peforming western detection protocols on midi sized membranes The iBind Flex Mini Insert is designed for peforming western detection protocols on up to two mini sized membranes Each row of the Mini Insert consists of two wells Lane 1 2 Well Position The iBind Flex Multi Strip Insert is designed for peforming western detection protocols on up to six vertically cut membrane strips Rows 2 and 4 consist of two wells each while rows 1 and 3 consist of six wells each Lane 1 2 3 4 5 6 Well Position IBind Flex Western Device User Guid
30. ts or keratin proteins when handling membranes Always handle membranes around the edges Non specific binding Primary antibody too Follow the supplier s recommended dilution or concentrated determine the optimum concentration by dot blotting Insufficient removal of Follow instructions for membrane preparation SDS weakly bound proteins from before immunodetection pages 12 13 membrane after blotting Affinity of the primary antibody for Check with protein standard manufacturer for the protein standards homologies with primary antibody Improper preparation of iBind Prepare 1X iBind Flex iBind Flex FD Flex iBind Flex FD Solution Solutions as directed pages 12 13 IBind Flex Western Device User Guide 29 Poor or incomplete transfer Refer to Western Blotting instructions IM 9051 and repeat blot After blotting stain Weak or no signal membrane to measure transfer efficiency Use positive control and or molecular weight marker Follow instructions for pre wetting the membrane Primary antibody concentration Follow the supplier s recommended dilution or too low determine the optimum concentration by dot blotting Inactive primary antibody Determine activity by performing a dot blot Low affinity of primary antibody to Obtain a higher affinity primary antibody antigen Contaminated secondary antibody Wear gloves at all times and keep bottles solution tightly capped when not in use Use only
Download Pdf Manuals
Related Search