iBlot® Western Detection Kit
Contents
1. 16X 6mL Deionized water 90 mL For regular sized membranes prepare enough 1X Wash Solution for at least four washes Adjust the volume of 1X Wash Solution according to the size of the dish used for washing the membrane Prepare an amount of Antibody Diluent Mix appropriate for the size of the membrane to be probed immediately before use Scale the volumes accordingly if performing immunodetection on multiple membranes Reagent Mini Regular Antibody Diluent Additive 4 5 mL 9mL Antibody Diluent Solution 10 5 mL 21 mL Total volume 15 mL 30 mL The same Antibody Diluent Additive is used for preparing solutions for PVDF and nitrocellulose membranes Continued on next page Preparing Solutions Continued Primary Antibody Concentration Preparing Primary Antibody Solutions Since protein immunodetection with iBlot Western Detection Kits are performed over a short period of time we recommend that you prepare dilutions of primary and secondary antibodies immediately before the procedure is started The concentration of the primary antibody can affect detection sensitivity and background Antibody solutions that are too dilute result in weak or no signal whereas overly concentrated solutions cause high background or non specific binding We recommend using twice the concentration of primary antibody required for a standard immunodetection e g if you usually dilute a primary antibody 1 5 000 use a dilution of 1 2 52. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 28 invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
3. membrane surface while adding the substrate Make sure the membrane is covered for the duration of the reaction Allow the reaction to develop for 5 minutes Blot any excess Chemiluminescent Substrate solution from the membrane by placing the membrane on filter paper with protein side facing up Do not allow the membrane to dry out Cover the membrane with plastic wrap to prepare a membrane sandwich for luminography Expose an X ray film to the membrane sandwich for 1 second to several minutes or image with an appropriate CCD camera Western Detection using the iBlot Dry Transfer System Continued Chromogenic Perform chromogenic development with the supplied Detection Chromogen Color development is complete in 1 60 minutes 1 Place the membrane with protein side facing up in a plastic tray supplied in the kit for mini sized membranes Do not allow the membrane to dry out Cover the blot with Chromogenic Substrate as follows Reagent Mini Regular Chromogen 5ml 10 ml Note If yellow precipitate forms after 3 minutes decant the solution wash briefly in deionized water and restart from step 1 Incubate with shaking until the desired purple band intensity is achieved on the membrane Do not incubate for more than 1 hour Decant solution Stop the reaction by rinsing membrane briefly with 20 ml of distilled water for 2 minutes and decanting the wash Repeat 2 minute water rinse twice Note Stop the reaction with r
4. tray or on other Spacers Uncut matrices can be used when membrane strips are treated under identical conditions e g performing the blocking step and using identical primary antibody conditions When using an uncut matrix place the membrane strips on the Bottom Stack with enough room between them to accommodate spacers in later steps Use cut matrices see page 21 and add spacers to the stack after the primary antibody step see page 16 Step 22 when testing different secondary antibody conditions Continued on next page Using Assay Spacers Continued Preparing the e Cut the antibody matrix using sharp clean scissors so Antibody Matrix that the pieces completely cover the membrane surface without overlapping other matrices or the Spacers e Apply blocking and antibody solutions in a volume that will completely wet but not soak the matrix see page 13 Step 3 page 15 Step 14 and page 16 Step 21 Using the e For all rolling steps do not roll over the spacer with the Blotting Roller hand held Blotting Roller e Assay Spacers are reusable Rinse with water after each use 21 Troubleshooting Introduction Review the information below to troubleshoot your experiments using the iBlot Gel Transfer Device and iBlot Gel Transfer Stacks Observation Cause Solution High background Nitrocellulose membrane not completely wetted Membrane is contaminated Follow instructions for pre wetti
5. 0 Pak iBlot Western Detection Chemiluminescent IB7210 02 Kit anti Rabbit Mini 10 Pak iBlot Western Detection Chromogenic Kit anti Mouse Regular 10 Pak IB7310 01 iBlot Western Detection Chromogenic Kit anti Mouse Mini 10 Pak IB7310 02 iBlot Western Detection Chromogenic Kit anti Rabbit Regular 10 Pak IB7410 01 iBlot Western Detection Chromogenic Kit anti Rabbit Mini 10 Pak IB7410 02 iBlot Western Detection Stacks Regular 10 Pak IB7010 01 iBlot Western Detection Stacks Mini 10 Pak IB7010 02 Intended Use For research use only Not intended for human or animal diagnostic or therapeutic uses Shipping and The iBlot Western Detection Kit is shipped as two boxes Storage The iBlot Western Detection Kit Stack Box is shipped and stored at room temperature The iBlot Western Detection Kit Reagent Box is shipped on blue ice and stored at 4 C Continued on next page Kit Contents and Storage Continued Stack Box Components Reagent Box Components The iBlot Western Detection Kit Stack Box contains enough transfer stacks for 10 mini sized blots 8 cm x 8 cm Mini or 10 regular sized blots 13 5 cm x 8 cm Regular Stack Box Components Mini Regular iBlot Western Detection Stack Bottom 10 10 iBlot Western Detection Stack Top 10 10 iBlot Disposable Sponge 10 10 iBlot Western Detection Antibody 30 30 Matr
6. 00 for the iBlot Western Detection protocol Note The total amount of primary antibody used is similar to that of a standard immunodetection protocol since only half as much volume is applied in the iBlot Western Detection protocol 1 Prepare the Primary Antibody Solution in a clean tube just prior to starting the immunodetection protocol as described below Reagent Mini Regular Antibody Diluent Mix 3 5 5 mL 7 10 mL Primary Antibody See Primary Antibody Concentration above 2 Mixthe solution well Continued on next page Preparing Solutions Continued Secondary Antibody Concentration Secondary Antibody Concentration for Chemi luminescent Detection Secondary Antibody Concentration for Chromo genic Detection 10 Unless the optimal secondary antibody concentration is already determined perform initial trials to find the optimal antibody concentration for your experiment Dilute the Secondary Antibody from the kit according to the directions below as a starting point For details on testing different antibody concentrations on multiple strips by using Assay Spacers refer to page 20 Note The antibody supplied in the kit is pre diluted 1 10 so that performing a 1 500 dilution as directed in the table below results in a final antibody concentration that is equivalent to a 1 5 000 dilution Dilute two aliquots of the secondary antibody according to the type of membrane you are using to determine the
7. cure the latch Press the Start Stop button to start the second 1 antibody step The two horizontal bars stop flashing While the iBlot Device runs apply the secondary antibody solution described on page 11 onto a new matrix Mini Regular 3 5 5 mL 7 10 mL After 3 minutes the device stops The end of the 1 antibody step is indicated by beeping and a flashing green light Open the lid leaving the Sponge in place Three flashing horizontal bars appear in the display between the program number and the time in the display Remove the Top Stack and set it aside in the red tray for re use in the next step Discard the used primary antibody matrix Use forceps to place the new matrix with the secondary antibody onto the membrane Remove any bubbles with the Blotting Roller Return the Top Stack to its position over the matrix Remove any air bubbles with the Blotting Roller Close the lid and secure the latch Press the Start Stop button to start the third 2 antibody step The three horizontal bars stop flashing After 3 minutes the device stops The end of the run is indicated by beeping and a flashing red light Continued on next page Western Detection using the iBlot Dry Transfer System Continued Disassembling 1 At the end of the transfer procedure open the lid and the Stack discard the sponge 2 Disassemble the sandwich and discard the used matrix Top Stack and Bottom Stack 3 Turn
8. eagent grade water do not use tap water buffer or acid Buffer or tap water can cause fading and acid turns the bands yellow Air dry the membrane on a clean piece of filter paper and record an image of the blot Store membrane protected from light to prevent band fading Bands remain visible for years when protected from light 19 Using Assay Spacers Guidelines for Using Assay Spacers Placing Membranes and Assay Spacers Using Assay Spacers for Secondary Antibody Optimization 20 Detection can be performed on multiple membrane strips in a single stack Stack assembly proceeds as described in the standard protocol page 13 with the following changes e Assay spacers can be placed vertically see picture below horizontally or as a combination of both e Divide transferred membranes into multiple strips equal to or less in surface area than the type of stack being used for western detection e To prevent cross contamination e g when using different antibodies for each strip use the supplied Assay Spacers to create a barrier between the strips e Membrane strips may need trimming to accommodate multiple strips and spacers in the same transfer stack e Place the cut membrane strips over the surface of the Bottom Stack see page 13 Step 6 Place the Assay Spacer between membrane strips using forceps or a gloved hand Leave a boundary region around membrane Do not overlap Spacers on the plastic rim of the
9. ibody Diluent Mix see page 8 Match gel separation range to size of protein being transferred 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 Continued on next page 23 Troubleshooting Continued Observation Cause Solution Non Specific Binding Membrane contaminated by fingerprints or keratin proteins Wear clean gloves at all times and use forceps when handling membranes Always handle membranes around the edges Concentrated secondary antibody used Make sure the secondary antibody is diluted as described on page 11 If the background remains high but with strong band intensity decrease the concentration of the secondary antibody Concentrated Primary antibody used Decrease the concentration of the primary antibody Affinity of the primary antibody for the protein standards Check with the protein standard manufacturer for homologies with primary antibody Error 2 Message Displayed Short circuit or current exceeding limits of device Open the lid for 15 seconds to allow the system to cool down Close the lid and resume the run by pressing the Start Stop button If problem persists contact Technical Support see page 26 Antibody Diluent Additive in the Antib
10. invitrogen by technologies iBlot Western Detection Kit For chemiluminescent and chromogenic detection of proteins on PVDF or nitrocellulose membranes Catalog nos IB7110 01 IB7110 02 IB7210 01 IB7210 02 IB7310 01 IB7310 02 IB7410 01 IB7410 02 IB7010 01 IB7010 02 Rev Date 18 June 2010 Part no 100009812 MANO0002735 Contents Kit Contents and Storages ege eisa e e ERER ERE rer EE REESE 3 LHteniu 5 About the System tenente 5 Experimental Overview sssesssssssseeeeeeneneeneneenee tenen 6 Lope 7 General Guidelines inde bee eer eei eei 7 Preparing Solutions in nenei n irn o ear i Ei 8 Western Detection using the iBlot Dry Transfer System 12 Using Assay Spacers sssssssssssssssseenenneneneeeneneneene eene 20 Troubleshootrig 5 ena peee bet en entendi 22 Por C I 25 Accessory Products s ise esee noie tenente diea 25 Technical Support eee ete ete eet eee tit eene en 26 Purchaser Notification stiga e a E a E E a E 27 Kit Contents and Storage This manual is supplied with the following products Types of Kits Product Cat no iBlot Western Detection Chemiluminescent IB7110 01 Kit anti Mouse Regular 10 Pak iBlot Western Detection Chemiluminescent IB7110 02 Kit anti Mouse Mini 10 Pak iBlot Western Detection Chemiluminescent IB7210 01 Kit anti Rabbit Regular 1
11. ix iBlot Western Detection Transparent 40 40 Sheet iBlot Western Detection Assay Spacer 2 4 Catalog no s S a s s e e e amp ls amp lselsel sellsels s ga age e pe 44 qt T EIEIRISISIR IRIE Component Oo 5 Oo 5 Oo o 5 S Wash Solution 16X 100 ml 21 2 1 2 1 2 1 Antibody Diluent Solution 3 213 l2 3 21l312 70 ml Diluent Additive 45 ml 2 1 2 1 2 1 2 1 Chemiluminescent 1 1 Substrate 30 ml Chemiluminescent 1 1 Substrate 60 ml Chemiluminescent 1 1 Enhancer 1 5 ml Chemiluminescent 1 1 Enhancer 2 5 ml Chromogen 100 ml 1 1 Chromogen 250 ml 2 SS exem UE 43 Anti Mouse 2 Antibody 211l l l2l1l l pre diluted 1 10 500 ul Anti Rabbit 2 Antibody l12111 1l21 1 pre diluted 1 10 500 ul Grid Dish Tray 2 LESE Dp 2 127 2 11 2 Introduction About the System System The iBlot Western Detection Kit consists of iBlot detection Description stacks and reagents that allow you to quickly perform immunodetection of transferred proteins on nitrocellulose or PVDF membranes using the iBlot Gel Transfer Device The iBlot system applies an electric field to transfer charged proteins primary and secondary antibodies towards a target membrane to accelerate the rate at which antibodies meet their antigens Detection is based on the use of specific secondary antibodies anti mouse anti rabbit conjugated to alkaline phosphatase and subsequent chemiluminescent development C
12. lose the lid and secure the latch The 12 red light is on indicating a closed circuit Select program P9 This program is a 3 step program for the iBlot Western Detection protocol The complete program runs for 8 minutes and cannot be modified Press the Start Stop button The red light changes to green A horizontal bar is displayed between the program number and the time in the display Do not turn off the iBlot Device or Bar appears here change programs at any step during program P9 While the iBlot Device runs apply the BA primary antibody solution described on page 9 onto a new matrix Mini Regular 3 5 5 mL 7 10 mL The first blocking step ends after 2 minutes and is indicated by beeping and a flashing green light Open the lid leaving the Sponge in place Two flashing horizontal bars appear between the program number and the time in the display Remove the Top Stack and set it aside in the red tray for re use in the next step Discard the used blocking soloution matrix Use forceps to place the new matrix with the primary antibody onto the membrane Remove any bubbles with the Blotting Roller Return the Top Stack to its position over the matrix Remove any air bubbles with the Blotting Roller Continued on next page 15 Western Detection using the iBlot Dry Transfer System Continued 19 20 21 22 23 24 25 26 27 28 16 Close the lid and se
13. lulose membrane Nitrocellulose membrane not completely wetted or PVDF membrane not completely reactivated Secondary antibody concentration too low Primary antibody concentration too low Inactive primary antibody Low Affinity of primary antibody to antigen Sample improperly prepared antigenicity weakened or destroyed Sample too dilute Blots are too old Incorrect ratio between Diluent Additive and Antibody Diluent Solution Protein of interest ran off the gel Poor retention of proteins Solution Check transfer conditions and repeat blot Use positive control and or molecular weight marker Add Enhancer to Chemiluminescent Substrate for nitrocellulose membranes Follow instructions for pre wetting or reactivating the membrane described on page 12 Use the secondary antibody concentrations described on page 10 Use twice the concentration of primary antibody required for a standard immunodetection If the signal is still low and the background is not high increase the concentration Determine activity by performing a dot blot or other methods Obtain a higher affinity primary antibody SDS and reducing agents may interfere with some antibody antigen affinities Load a higher concentration or amount of protein onto the gel Protein may have broken down over time Use freshly prepared blots Make sure the proper amount of Diluent Additive is used for preparation of Ant
14. lution with Diluent Additive described on page 8 evenly on the matrix with a clean pipette for the blocking step Mini Regular 3 5 5 mL 7 10 mL 4 Remove the sealing from the iBlot Western Detection Bottom Stack Leave the stack in the transparent plastic tray 5 Place the plastic tray containing the Bottom Stack directly on the blotting surface Align the tray with the gel barrier on the right 6 Use forceps to place the pre wetted membrane on the Bottom Stack with the protein side facing up Remove any bubbles using the Blotting Roller Note The Blotting Roller is used several times throughout this protocol and should be washed between each step Continued on next page 13 Western Detection using the iBlot Dry Transfer System Continued 7 Use forceps to place the matrix soaked with Antibody Diluent Mix Step 3 onto the membrane 8 Remove any bubbles using the Blotting Roller 9 Remove the sealing from the iBlot Western Detection Top Stack Keep the red plastic tray for Step 16 10 Remove the Top Stack from the tray and place it over the green matrix with the electrode side facing up Remove any bubbles with the Blotting Roller 11 Position the iBlot Disposable Sponge so the metal contact is at the upper right corner of the lid Continued on next page 14 Western Detection using the iBlot Dry Transfer System Continued 12 13 14 15 16 17 18 C
15. m x 13 cm for regular sized iBlot Western Detection Stacks e Use twice the concentration of primary antibody required for a standard immunodetection e Usea single clean dish for each blot The container must be large enough to allow the membrane to be fully covered by solutions at all times Note Western Detection Stacks are supplied with two mini sized Grid Dish Trays e Avoid touching the surface of the membrane Wear clean gloves and handle the blot only with clean forceps e Work quickly to ensure membranes remain wet e Do not expose the substrate working solutions to intense light Short term exposure to laboratory light is not harmful to the substrates e Do not use Western Detection Stacks for protein transfer To increase the rate of success on the first trial we recommend running replicates of your sample on a single gel and preparing multiple strips for simultaneous detection see Using Assay Spacers page 20 using different secondary antibody concentrations on a single stack see page 10 Preparing Solutions Important Preparing Wash Solution Preparing Antibody Diluent Mix Use water free from alkaline phosphatase activity for making wash buffer and rinsing membranes Fresh ultra filtered water is preferred Autoclave or ultra filter stored water to remove alkaline phosphatase activity Prepare 96 mL of 1X Wash Solution for each mini sized membrane to be probed Reagent Volume Wash Solution
16. mation and special offers For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Tel 1 760 603 7200 Japanese Headquarters European Headquarters LOOP X Bldg 6F Inchinnan Business Park 3 9 15 Kaigan 3 Fountain Drive Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech supportGinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com SDS Certificate of Analysis 26 Safety Data Sheets SDSs are available at www invitrogen com sds The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Purchaser Notification Limited Warranty Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitr
17. ng the membrane Use only clean new membranes Wear clean gloves at all times and use forceps when handling membranes Incorrect ratio between Diluent Additive and Antibody Diluent Solution Make sure the proper amount of Diluent Additive is used for preparation of Antibody Diluent Mix see page 8 Blot is overdeveloped Follow recommended developing time or remove blot from substrate when signal to noise ratio is acceptable Incorrect program was used Use only program P9 for the iBlot Western Detection protocol Higher intrinsic background with PVDF membranes Switch to nitrocellulose membranes Enhancer added to substrate when using PVDF membrane Make sure Enhancer is not added to Chemiluminescent Substrate for PVDF membranes Insufficient washing Follow recommended number of washes In some cases it may be necessary to increase the number or duration of washes Concentrated secondary antibody used Make sure the secondary antibody is diluted as described on page 11 If the background remains high but with strong band intensity decrease the concentration of the secondary antibody Concentrated primary antibody used Decrease the concentration of the primary antibody 22 Continued on next page Troubleshooting Continued Observation Weak or No Signal Cause Poor or incomplete transfer Enhancer not added to substrate when using nitrocel
18. ody Diluent Mix exceeds 30 causing increased current and heat Make sure the Antibody Diluent Mix is prepared as described on page 8 24 Appendix Accessory Products Additional The following additional products including a variety of Products reagents for western blotting and western detection are available from Invitrogen for use with the iBlot Gel Transfer Device For more details on these products visit our website at www invitrogen com or contact Technical Support see page 26 Product Quantity Catalog no iBlot Gel Transfer Device 1 unit IB1001 iBlot Western Detection Stacks Regular 10 pak IB6010 01 iBlot Western Detection Stacks Mini 10 pak IB6010 02 iBlot Gel Transfer Stack Nitrocellulose Regular 10 pak IB3010 01 iBlot Gel Transfer Stack PVDF Regular 10 pak IB4010 01 iBlot Gel Transfer Stack Nitrocellulose Mini 10 pak IB3010 02 iBlot Gel Transfer Stack PVDF Mini 10 pak 1B4010 02 Blotting Roller 1 LC2100 A variety of antibodies are available from Invitrogen For more details visit www invitrogen com antibodies 25 Technical Support Web Resources Contact Us Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product infor
19. off the iBlot Device 4 Proceed to Washing the Membrane Washing the Wash volumes are described below for mini sized Membrane membranes For regular sized membranes adjust volumes according to the size of the container being used for the wash 1 Place the membrane into a dish a mini sized dish is provided in the kit containing 20 mL of 1X Wash Solution page 8 2 Rinsethe membrane briefly and then discard the solution 3 Wash the membrane for 5 minutes with 20 mL of 1X Wash Solution and discard the solution Repeat this wash step two more times 4 Rinse the membrane with 20 mL of deionized water and then decant Repeat this wash step once 5 Proceed to the Chemiluminescent Detection or Chromogenic Detection step 17 Western Detection using the iBlot Dry Transfer System Continued Chemi luminescent Detection 18 Prepare the following amount of Chemiluminescent Substrate per membrane Nitrocellulose Membrane Mini Regular Chemiluminescent Substrate 3mL 6 mL Chemiluminescent Enhancer 0 125 mL 0 25 mL PVDF Membrane Mini Regular Chemiluminescent Substrate 3mL 6 mL Mix well Do not add Chemiluminescent Enhancer for PVDF membranes Place the membrane with protein side facing up on a sheet of Transparency plastic iBlot Western Detection Transparent Sheet supplied in the kit Do not allow the membrane to dry out Cover the membrane with 3 mL of Chemiluminescent Substrate in an even application Do not touch the
20. ogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Continued on next page 27 Purcha
21. opper Cathode iBlot Western Detection Stack Top iBlot Western 1 Detection Antibod y Matrix PVDF or NC membrane with transferred protein i Cu gt Cu 2e xv iBlot Western Detection Stack Copper Anode Bottom Features e Compatible with proteins transferred by wet semi wet semi dry or iBlot dry blotting methods e Rapid binding of antibodies to antigens completed in three minutes Experimental Overview Workflow Western detection can be performed on one regular sized membrane 13 5 cm x 8 cm 1 2 mini sized membranes 8 cm x 8 cm or multiple membrane strips using supplied spacers to prevent cross contamination Before Starting 1 Transfer proteins from gel to membrane 2 Wash the membrane in deionized water 3 Prepare wash and antibody solutions Blocking Step 4 Apply the Antbody Diluent Mix to a matrix 5 Set the iBlot device to program P9 consists of 3 steps for a total of 8 minutes and assemble the Western Detection Stack Bottom Stack membrane antibody matrix with Antbody Diluent Mix Top Stack 6 Block the membrane program P9 step 1 for 2 minutes Primary Antibody Step 7 Apply the primary antibody solution on a new matrix 8 Remove and discard used blocking solution matrix 9 Reassemble the Western Detection Stack with primary antibody matrix and perform the primary antibody step program P9 step 2 for 3 minutes Secondary Antibody Step 10 A
22. optimal concentration Nitrocellulose Membrane PVDF Membrane 1 250 1 500 1 500 1 1 000 If necessary modify the concentration for a second trial based on the initial results as follows Initial Result Nitrocellulose PVDF Membrane Membrane Low Signal 1 100 1 250 High background 1 1 000 with strong signal Note Increasing the secondary antibody concentration may result in increased background Dilute two aliquots of the secondary antibody according to the type of membrane you are using Nitrocellulose Membrane PVDF Membrane 1 100 1 100 1 250 1 250 Continued on next page Preparing Solutions Continued Preparing Secondary Antibody Solutions Prepare the Secondary Antibody Solution in a clean tube as described below Use the secondary antibody supplied in the kit Do not use antibodies from a different kit or other supplier Reagent Mini Regular Antibody Diluent Mix 3 5 5 mL 7 10 mL Secondary Antibody See Secondary Antibody Concentration page 10 Mix the solution well 11 Western Detection using the iBlot Dry Transfer System Before Starting Transferring Proteins Important Preparing Membranes Materials Required 12 Ensure that the iBlot Device is set to program P9 see page 7 for the western detection protocol Prepare solutions before starting the protocol see page 8 Wet membranes if they are dry see below Blot proteins onto nit
23. pply the secondary antibody solution on a new matrix 11 Remove and discard used primary antibody matrix 12 Reassemble the Western Detection Stack with secondary antibody matrix and perform the secondary antibody step program P9 step 3 for 3 minutes 13 Disassemble the stack and rinse the membrane in Wash Solution 14 Wash the membrane in Wash Solution three times 15 Prepare detection reagents 16 Add the appropriate detection substrate 17 Develop and visualize the blotted membrane Methods General Guidelines Firmware Requirements General Guidelines a RECO EN Nowe l Firmware version 2 9 5 or higher with program P9 is required to use the iBlot Western Detection Stacks The iBlot firmware version is displayed on the screen upon powering the device on For users of the iBlot Device with older firmware versions lacking program P9 download new iBlot firmware with program P9 version 2 9 5 or higher at www invitrogen com iblot Do not use iBlot Western Detection Stacks if you cannot run program P9 Note Program P9 is a 3 step program for the iBlot Western Detection protocol consisting of a blocking step 20V for 2 minutes a primary antibody step 5V for 3 minutes and a secondary antibody step 5V for 3 minutes The overall time of the program is 8 minutes and cannot be modified e Membranes should not exceed 8 cm x 8 cm for mini sized iBlot Western Detection Stacks or 8 c
24. rocellulose or PVDF stacks with the iBlot Device or by standard wet semi wet or semi dry transfer methods appropriate for the protein to be detected After transfer rinse the membrane with water to remove any gel and transfer buffer components After transfer and rinsing membranes can be dried and stored for immunodetection at a later time Do not perform immunodetection on dry membranes Verify that membranes are wet before performing detection PVDF membranes dry quickly and must be reactivated with methanol prior to starting the protocol If starting the protocol with dried membranes re wet the membranes using the following steps Membrane Procedure Nitrocellulose Wet the nitrocellulose membrane with distilled water for 1 minute PVDF Reactivate the PVDF membrane in 100 methanol for 15 seconds and rinse twice with water Blotted membrane with antigen of interest Purified water free of alkaline phosphatase activity Clean tubes for preparing solutions Clean forceps for manipulating blotted membrane Orbital shaker capable of rotating at 1 revolution second 1X Wash buffer see page 8 Primary antibody diluted in Antibody Diluent see page 8 Continued on next page Western Detection using the iBlot Dry Transfer System Continued 1 Open the lid of the iBlot Gel Transfer Device Ensure the blotting surface is clean 2 Place the green antibody matrix on a Transparent Sheet 3 Apply Antibody Diluent So
25. ser Notification Continued Limited Use The purchase of this product conveys to the buyer the non Label License transferable right to use the purchased amount of the product No 5 Invitrogen and components of the product in research conducted by the Technology buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research
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