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Transfer Stack

Method

Volts

Run Time

Regular transfer

stack
(1 midi gel or two

mini gels)

P0, P3

20–25

8–10 minutes

Regular transfer

stack
(E-PAGE

™

48 or 96

gel)

P0, P3

20–25

8–10 minutes

Mini transfer stack
(one mini gel)

P0, P3

20–25

8–10 minutes

You may need to optimize the blotting parameters (volts or time) based on your initial

results (See “Optimizing blotting“ on page 49 for details). This may include:

• Increasing or decreasing the transfer time.
• Performing an ethanol equilibration step prior to transfer can improve overall

efficiency of protein transfer.

Chapter 1

Product information

Description of methods

iBlot

™

2 Dry Blotting System User Guide

Содержание iBlot 2

Страница 1: ...e Only Not for use in diagnostic procedures iBlot 2 Dry Blotting System USER GUIDE For dry electroblotting of proteins from mini midi and E PAGE gels Catalog Number IB21001 Publication Number MAN00091...

Страница 2: ...ical contacts section Converted to CCMS D 0 15 March 2017 Addition of detailed information on proper method of closing device lid Important Licensing Information These products may be covered by one o...

Страница 3: ...ry Blotting System 9 Features 10 System components 10 System overview 10 Description of parts 12 iBlot 2 Gel Transfer Device 12 Blotting roller 13 Stylus 14 Power cord 14 Power adapters 14 iBlot 2 Tra...

Страница 4: ...gel types 26 Using the iBlot 2 Gel Transfer Device 26 Introduction 26 Materials needed 26 Selecting a method 27 Removing the gel 28 General guidelines 28 Assembling the iBlot 2 Transfer Stack 29 Perf...

Страница 5: ...ot 2 Transfer Stacks 57 Additional products 57 Precast gels and premade buffers 58 APPENDIX G Safety 59 Instrument safety 59 General 59 Physical injury 59 Electrical safety 60 Cleaning and decontamina...

Страница 6: ...way that may cause personal injury or property damage You are responsible if the product is used for any intention other than its designated purpose or in disregard of Thermo Fisher Scientific instruc...

Страница 7: ...rements Room ventilation Do not use if device becomes cracked or broken in any area The product may be installed only under the conditions and in the positions specified by Thermo Fisher Scientific Fo...

Страница 8: ...ing transit File any damage claims with the carrier The warranty does not cover in transit damage Remove the roller and stylus from the Styrofoam packaging Remove the device from the Styrofoam packagi...

Страница 9: ...lot 2 Transfer Stacks results in a shortened distance between electrodes high field strength and high currents to reduce transfer times when blotting proteins onto membranes Western blotting of protei...

Страница 10: ...2 Gel Transfer Device on page 12 for details iBlot 2 Transfer Stacks The iBlot 2 Transfer Stacks are disposable stacks that have integrated PVDF or nitrocellulose transfer membranes to perform dry bl...

Страница 11: ...s This format eliminates the need for premade buffers or soaked filter paper and minimizes handling that can lead to inconsistent performance The copper anode does not generate oxygen gas as a result...

Страница 12: ...of methods on page 23 for details on each Method IMPORTANT When installing the iBlot 2 Gel Transfer Device make sure it is placed on a level surface Keep the area around the device clear to ensure pr...

Страница 13: ...on the stack surface when the lid is closed Control panel The Touch Screen Control Panel is a LCD display allowing the user to select Methods and control the device See Control panel of the iBlot 2 G...

Страница 14: ...tion Rear view on page 9 before attaching the power cord Attach the power cord to the AC inlet of the device first and then to the electrical outlet Use only properly grounded AC outlets and power cor...

Страница 15: ...Stack Do not use iBlot Transfer Stacks in the iBlot 2 Gel Transfer Device or mix components between iBlot Transfer Stacks and iBlot 2 Transfer Stacks Use iBlot 2 Transfer Stacks only for their design...

Страница 16: ...the support for assembling the transfer stacks with the gel The nitrocellulose 0 2 m and PVDF 0 2 m membranes do not require any pretreatment before use and minimize protein blow through during the i...

Страница 17: ...e 55 for dimensions for efficient blotting of mini and midi gels Do not use the iBlot Filter Paper for blotting E PAGE gels Note Failure to use the iBlot Filter Paper during blotting of mini or midi g...

Страница 18: ...een 3 OR skip the tutorial to go to the Home Screen The control panel is a touch screen display allowing 1 Running the Method that was last used on the device Note This icon will not appear until afte...

Страница 19: ...tep tutorial for the iBlot 2 Gel Transfer Device Write logs to a USB device View application notes Upgrade firmware Reset the iBlot 2 Gel Transfer Device to factory settings iBlot 2 Gel Transfer Devic...

Страница 20: ...tions screen The Tutorial button provides step by step instructions for assembling and running a transfer stack on the iBlot 2 Gel Transfer Device If following the tutorial to assemble a stack in real...

Страница 21: ...rage device will appear Touch OK to start the transfer after inserting the USB storage device 3 Do not remove the USB storage device until the Options screen is displayed again 4 The files are saved t...

Страница 22: ...in place a prompt to insert a USB storage device will appear 5 Touch OK to start the update Do not remove the USB storage device until the Options screen is displayed again showing the new firmware ve...

Страница 23: ...me setting for a selected Method The Run Time Limit is the maximum recommended run time for a selected Method The following parameters are recommended for transfer of proteins with molecular weights r...

Страница 24: ...25 8 10 minutes You may need to optimize the blotting parameters volts or time based on your initial results See Optimizing blotting on page 49 for details This may include Increasing or decreasing th...

Страница 25: ...orming blotting on page 33 5 Disassemble the iBlot 2 Transfer Stack Disassembling the iBlot 2 Transfer Stack on page 34 General guidelines To obtain the best results follow these recommendations Wear...

Страница 26: ...lycine Midi gels or equivalent 13 cm l 8 3 cm w 1 0 mm thick iBlot 2 Regular Transfer Stack Mini gels Bolt Bis Tris PLUS NuPAGE Bis Tris or Tris Acetate Tricine Tris Glycine Gels or equivalent 8 cm l...

Страница 27: ...sfer Device The fan in the device begins to run and the digital display turns on 2 Select the Method by touching Templates to access the desired Method see Control panel of the iBlot 2 Gel Transfer De...

Страница 28: ...format in the stacks without any need for pretreatment Do not treat the PVDF membrane with methanol as the PVDF membrane is preactivated prior to assembly with the transfer stack You may blot E PAGE g...

Страница 29: ...e latch Ensure the blotting surface is clean 2 Unseal the iBlot 2 Transfer Stack Assembling the iBlot 2 Transfer Stack Chapter 2 Protein transfer protocol Using the iBlot 2 Gel Transfer Device 2 iBlot...

Страница 30: ...may elevate and twist the tray or interfere with the contacts The electrical contacts on the tray should be aligned with the corresponding electrical contacts on the blotting surface of the iBlot 2 Ge...

Страница 31: ...iBlot 2 Regular Transfer Stack 10 1 mini gel on an iBlot 2 Mini Transfer Stack 11 Use the Blotting Roller to remove any air bubbles between the gel and the membrane Chapter 2 Protein transfer protocol...

Страница 32: ...Note For E PAGE gels there is no need to use a filter paper 13 Place the presoaked iBlot Filter Paper on the pre run gel Use the Blotting Roller to remove any air bubbles between the filter paper and...

Страница 33: ...g within 10 15 minutes of assembling the stacks with the gel 1 Gently close the iBlot 2 Gel Transfer Device lid by pressing down with two hands on the sides of the lid Make sure the latch is secure Do...

Страница 34: ...shown below Remove the transfer membrane from the stack and proceed with the blocking procedure or stain the membrane see for details Note If you are using PVDF membranes place the membrane immediate...

Страница 35: ...ready for another run no cooling period is required If you are not using the device turn off the power switch located on the back of the iBlot 2 Gel Transfer Device IMPORTANT Do not reuse the iBlot 2...

Страница 36: ...nsfer Device for specific applications or fine tuning transfer conditions 1 Select New Method 2 Select the number of steps to be included in the new Method using the buttons Up to 10 steps can be adde...

Страница 37: ...t Done after programming the final step and the entire Method is displayed Select Next and proceed to to Save a Custom Method Save a custom method on page 38 Note The Edit button can be used at this p...

Страница 38: ...keyboard to enter a name for the new Method 2 Save the method 3 The new Method will appear in the list of Saved Methods Save a custom method Chapter 3 Custom methods Creating custom methods 3 38 iBlo...

Страница 39: ...fine tuning transfer conditions using a pre set template or previously saved Method as a starting point 1 Select Templates 2 Select a Method from the list on the Preset Templates or Saved Methods scr...

Страница 40: ...steps on page 41 1 Select voltage or time fields in any given step while in Edit Mode and a keyboard will appear 2 Enter desired values for voltage or time and select Done 3 After editing the Edit Me...

Страница 41: ...ted and proceed to to Save a Custom Method Save a custom method on page 43 1 Select Manage Steps while in Edit Mode Add remove steps Chapter 3 Custom methods Creating custom methods from a template 3...

Страница 42: ...a step or the icon to remove a step 4 If a step is added a screen appears to enter the desired voltage and time for that step After the values are added select Done to return to the Edit Method screen...

Страница 43: ...een and proceed to to Save a Custom Method Save a custom method on page 43 1 Click Save to save the method Save a custom method Chapter 3 Custom methods Creating custom methods from a template 3 iBlot...

Страница 44: ...reen and use the keyboard to enter in the characters Touch OK when done 3 The new Method will now appear under Saved Methods icon button Chapter 3 Custom methods Creating custom methods from a templat...

Страница 45: ...ce Plastic separator was not removed when assembling stack Make sure that the plastic separator is removed from the stack see Assembling the iBlot 2 Transfer Stack on page 29 The metal safety contacts...

Страница 46: ...he package High molecular weight proteins remain in the gel indicated by staining of the gel after transfer Incorrect method or transfer conditions used Note It is normal for some proteins to remain i...

Страница 47: ...ization based on your initial results Corrosion of the Top Stack Incorrect placement of the Top Stack Be sure the Top Stack is placed correctly with the copper electrode facing up Avoid placing the To...

Страница 48: ...for a few seconds then rinse the membrane thoroughly with deionized water to remove methanol To stain membranes after blotting use any method of staining for total protein visualization such as Coomas...

Страница 49: ...the recommended run time limit indicated for each Method Description of methods on page 23 Proteins 150 kDa migrate more slowly and require more time to transfer If your protein of interest is in this...

Страница 50: ...uses and electrical contacts Disconnect the unit from the power supply by removing the power cord before performing either of these maintenance procedures For any other repairs and service contact Tec...

Страница 51: ...s with a damp cloth after each use To replace an electrical contact a 1 Phillips Screwdriver is required 1 Make sure the device power is OFF then open the lid of the iBlot 2 Gel Transfer Device 2 Hold...

Страница 52: ...he Round Bumper from the Lid Discard the Round Bumper 5 Open the bag of the Electrode Replacement Kit Cat no IB28001 and remove all of the parts Appendix D Maintenance Replacing electrical contacts D...

Страница 53: ...through the hole in one of the contacts Position the contact screw onto the Contact 1 location Note the contact has a small orientation notch which matches a bump in the Lid This is to ensure proper...

Страница 54: ...in one of the contacts Position the contact screw onto the Contact 2 location Turn the screw with a screwdriver until it is hand tight Appendix D Maintenance Replacing electrical contacts D 54 iBlot...

Страница 55: ...6 3A Slo Blo 5x20 250V Littelfuse 021806 3HXP The iBlot 2 Gel Transfer Device is impervious to alcohol acid HCl alkali NaOH but not compatible with acetone dimethyl sulfoxide and acetic acid iBlot 2...

Страница 56: ...rescence Plastic Tray 16 8 cm 10 3 cm 1 7 cm wide copper contact iBlot 2 Absorbent Pad Dimensions 15 cm l 9 5 cm w 1 1 cm thick Material Gray Melamine Metal Contact Aluminum iBlot Filter Paper Regular...

Страница 57: ...002 Blotting Roller 1 unit LC2100 Additional products Additional reagents that may be used for electrophoresis of proteins are available at thermofisher com Ordering information is provided below For...

Страница 58: ...tandard 250 L LC5602 Novex Reversible Membrane Protein Stain Kit 1 kit IB7710 SYPRO Ruby Protein Blot Stain 200 mL S 11791 Precast gels and premade buffers A large variety of precast gels including Bo...

Страница 59: ...s read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection and so on To obtain SDSs see the Documentation and Support se...

Страница 60: ...he instrument AVERTISSEMENT Veiller utiliser une alimentation lectrique appropri e Pour garantir le fonctionnement de l instrument en toute s curit Brancher le syst me sur une prise lectrique correcte...

Страница 61: ...gents de nettoyage ou de d contamination susceptibles de r agir avec certaines parties de l appareil ou avec les mati res qu il contient et de constituer de ce fait un DANGER L instrument doit tre cor...

Страница 62: ...f WEEE Visit http www lifetechnologies com weee for collection and recycling options The Electrical Warning symbol denotes a risk of electrical shock hazards To avoid risk of injury from electric shoc...

Страница 63: ...19 EU European Union WEEE Directive Waste electrical and electronic equipment Directive 2011 65 EU European Union RoHS Directive Restriction of hazardous substances in electrical and electronic equipm...

Страница 64: ...lysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according...

Страница 65: ...tions peuvent s appliquer leur limination Biological hazard safety WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the pot...

Страница 66: ...and protocols Certificates of Analysis Safety Data Sheets SDSs also known as MSDSs Note For SDSs for reagents and chemicals from other manufacturers contact the manufacturer Limited product warranty L...

Страница 67: ......

Страница 68: ...thermofisher com support thermofisher com askaquestion thermofisher com 19 February 2019...

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