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View additional product information for iBlot® Gel Transfer Device - FAQs (IB1001)
51 product FAQs found
Yes, all original iBlot stacks are still available for purchase. You can find them in the original iBlot Gel Transfer Device Reagents and Resources (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/iblot-dry-blotting-system/reagents-resources-original-iblot-gel-transfer-device.html). However, please note that they are not compatible with the iBlot 2 Gel transfer Device.
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No. Do not use iBlot Transfer Stacks in the iBlot 2 Gel Transfer Device, and do not mix components between iBlot Transfer Stacks and iBlot 2 Transfer Stacks.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from gel to membrane. We recommend extending the transfer time by 8 to 10 minutes for optimal transfer of proteins >150 kDa using the iBlot Dry Blotting System.
To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE Invitrogen 3 - 8% Tris-acetate gels for electrophoresis. We have an application note available, titled “Transferring Large and Small Proteins Using the iBlot Dry Blotting System”. To download the PDF, see the iBlot Reagents Resources page. (http://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/western-blotting/western-blot-transfer/iblot-dry-blotting-system/original-iblot-resources.html)
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This may be a result of insufficient blocking or nonspecific binding. We suggest trying our WesternBreeze Blocker/Diluent (Cat. No. WB7050). We have been using it with good success. Additionally, you should optimize primary and secondary antibody concentrations as generally recommended for any new blotting technique. Many cases of high background can be resolved by further diluting one or both antibody preparations.
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In theory, you can replace the membrane provided in your iBlot Transfer Stack with any membrane that is compatible with western blotting. To do this, cut the alternative membrane to match the size of your gel, and wet the membrane. Then, either place the alternative membrane on top of the integrated membrane, or carefully remove the integrated membrane from the gel matrix with forceps and replace it with the new membrane. Note that we only support the use of iBlot Transfer Stacks when they are used with the provided instructions.
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The minimum guaranteed shelf life of iBlot Transfer Stacks is 2 months. Depending on when you purchase the transfer stack, shelf life will be 2–8 months. The expiration date is printed on the package for each stack.
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No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.
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No. The PVDF membrane comes preactivated. You just need to open the transfer stack with membrane, place the separation gel on top of the membrane, and apply one layer of moistened filter paper to run (the same as with the nitrocellulose stacks).
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The green discoloration is copper deposits from the iBlot Transfer Stacks, and it does not affect the results. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack. The current formulation of stacks minimizes the green discoloration.
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The plastic in the iBlot Dry Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.
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It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The iBlot Dry Blotting System has been tested to efficiently transfer protein from gels ranging in thickness from 1 mm to 3 mm. We have not tested gels thicker than 3 mm because they are rarely used for SDS-PAGE.
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Yes. While we would prefer that you use our devices, Bolt gels can also be transferred using devices from Bio-Rad, including the Mini Trans-Blot Cell, Trans-Blot SD Semi-Dry Transfer Cell, or Trans-Blot Turbo Transfer System.
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Small proteins (under 30 kDa) migrate more rapidly than large ones and hence, need less time to transfer from the matrix of the gel to the membrane. While P3 program for 7 minutes works well with most proteins, less time is needed for the transfer of smaller proteins with the iBlot device. We recommend using P3 program for less than 5-6 mins. Please refer to Transferring Large and Small Proteins Using the iBlot Dry Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/protein-expression/pdfs.par.87290.file.dat/iblot%2520small%2520and%2520large%2520protein%2520app%2520note.pdf).
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After staining with SimplyBlue SafeStain, use deionized water for the less strongly retained protein bands on the PVDF membrane.
Increasing methanol or ethanol concentrations up to 70% should destain any remaining bands. You can leave the membrane in the destain indefinitely.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This is most likely due to uneven current distribution during transfer, possible caused by bad electrodes. We recommend getting new electrodes (Cat. No. IB1002).
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This could happen due to the USB cable disconnecting during the updating process. Please try again with another computer and a different USB cable.
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The iBlot device must be connected directly to the computer for the firmware upgrade to work. Do not use a USB hub.
In order to make a loop-back test you first need to make sure that the drivers and COM port have been successfully installed.
1. With the USB Serial drivers installed, connect your iBlot device to your computer's USB port.
2. Check in Windows Device Manager (Start -> Control Panel -> System -> Hardware tab -> Device Manager) under Ports (COM & LTP) to see if the USB Serial Port (COM) has been successfully created. If the COM port is not listed in the Device Manager, it has not been successfully created. You can install the driver manually from http://www.ftdichip.com/Drivers/VCP.htm
3. If multiple devices are connected to one USB serial port, you may need to disconnect other devices.
If the above steps don't help, another simple option that may solve the issue would be to try a different USB cable.
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It is possible that the PVDF membrane got displaced during handling or shipment. The activated PVDF membrane is transparent, making it difficult to see. If it is not present on top of the stack, we recommend examining the aluminum seal of the Anode Stack (clear tray) to make sure that the PVDF membrane has not adhered to the seal. If the membrane has adhered to the seal, we suggest reactivating the membrane with pure methanol, then rinsing it well in distilled water, and carefully placing it on the stack. The performance will not be affected by the reactivation process.
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This is most likely due to the protein load being too high, as a result of which detection is not within the linear range. Since the immunodetection sensitivity is higher for dry blotting with the iBlot Gel Transfer Device than for semi-dry or wet blotting, we recommend that you decrease the protein load, use more diluted antibody, or perform detection for shorter time. You may need to perform some optimization based on your initial results.
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This is likely due to use of TBST buffers for washing. We recommend using PBST or WesternBreeze wash solutions.
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This is likely due to the PVDF membrane being dry or partially dry. Regions where PVDF membranes are dry appear whiter than places where the membrane is wet. Remove the membrane and reactivate in 100% methanol, and rinse in water before reapplying to the transfer stack.
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This indicates that a non-uniform electric field was created around the wells. Ensure that the well protrusions on the E-PAGE gel are properly flattened using the De-bubbling Roller. To ensure the best blotting results, we recommend using the De-bubbling Roller with E-PAGE gels. If you use the Blotting Roller with E-PAGE gels, be sure to follow the recommendations on page 22 of the manual (https://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf) to obtain good results.
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This could happen if the transfer time used is too long. We recommend reducing the transfer time by 30 second increments.
Note: Pre-stained markers are charged, so tend to blow-through more than regular proteins.
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This could happen if an incorrect voltage Method was used or if inappropriate transfer conditions were used. Make sure that the voltage Method and run time used is correct, based on the gel type, as described on page 13 in the manual.
For mini or midi gels:
- Perform an ethanol equilibration step as described on page 28 in the manual to improve transfer.
- Use a lower gel percentage to separate the high-molecular weight proteins.
- Increase the transfer time in 30-second increments.
For E-PAGE gels:
- Increase the transfer time in 30-second increments.
- Use Method P2 for 8 minutes.
Note: It is normal for some proteins to remain in the gel because some high-molecular weight proteins do not transfer completely using the iBlot Gel Transfer Device, compared to semi-wet transfer apparatus.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- Presence of air bubbles between the gel and the membrane preventing the transfer of proteins. Be sure to remove all air bubbles between the gel and membrane by using the De-bubbling Roller for E-PAGE gels or Blotting Roller for other gels.
- Expired or creased membranes used. Use the iBlot Gel Transfer Stacks before the expiration date printed on the package.
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This could be due to no current passing through or because an incorrect voltage Method was used. Make sure that the electrical circuit is complete and current is flowing through the device. Please check to make sure that the correct voltage Method is used, see page 13 in the manual (http://tools.thermofisher.com/content/sfs/manuals/iblotsystem_man.pdf).
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This happens if the membrane is trimmed to fit the gel size resulting in direct contact between the iBlot Cathode, Top and Anode Bottom stacks. Always maintain the membrane size identical to the transfer stack. Transfer quality is not affected by smaller gel size compared to the membrane.
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The green discoloration is due to copper ions from the iBlot Transfer Stacks being carried by liquids and deposited on the membrane. These deposits do not interfere with downstream processes. The stained regions can be cut away, but membrane washing typically results in their removal. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack.
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Longer transfer times result in the deposition of copper ions, causing the blue discoloration. Be sure to perform the transfer for the recommended time for each gel type.
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This is most likely due to incorrect placement of the top Stack. Be sure the iBlot Cathode Stack, Top is placed correctly with the copper electrode side facing up. Avoid placing the top stack in the inverted position.
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Here are possible causes and solutions:
- The iBlot Cathode Stack, Top is touching the copper electrode on the iBlot Anode Stack, Bottom: Open the lid and align the iBlot Cathode Stack, Top to the right. Continue the run by briefly pressing Start/Stop Button or restart the run by pressing and holding the Start/Stop Button.
- The layers are not aligned: Align the layers properly as described in the protocols. Ensure that the electrodes are in contact.
- Current is above 5.5 amp: Select a program with a lower voltage. Open the iBlot Lid and ensure the stacks are aligned properly. Close the lid and restart the run by subtracting the time already elapsed. Replace the iBlot Gel Transfer Stacks with fresh transfer stacks. Ensure an iBlot Filter Paper was used during blotting of mini- or midi-gels.
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This indicates an open electrical circuit during the run. It occurs if the lid opened during the run.
Close the lid and continue the run by briefly pressing the Start/Stop Button or restart the run by pressing and holding the Start/Stop Button.
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This indicates an incomplete electric circuit and here are possible causes and solutions:
- iBlot Disposable Sponge covers the metal contact or the metal contact on the sponge is on the left: Reinsert the iBlot Disposable Sponge such that the metal contact on the sponge is on the top right of the lid and is in contact with the electrode on the transfer stack.
- Incorrect position of the transfer stacks or improper assembly of the transfer stacks: Make sure the transfer stack is placed in the proper position in the blotting surface to allow proper contacts with the electrodes. Ensure the transfer stacks are assembled correctly; use the iBlot Anode Stack, Bottom first followed by the gel and iBlot Cathode Stack, Top.
- Incorrect position of the pull tab: Ensure the pull tab from the iBlot Cathode Stack, Top is towards the right of the assembly in the blotting surface.
- iBlot Anode Stack, Bottom placed on the device without the tray including the electrical contact: Do not remove the iBlot Anode Stack, Bottom from the tray during the assembly. The blotting is performed with the bottom stack in the plastic tray.
- iBlot Cathode Stack, Top placed on the device with the tray: Always remove the iBlot Cathode Stack, Top from the red plastic tray before placing the top stack on the assembly. Do not use the iBlot Cathode Stack, Top with the tray.
- The metal safety contacts in the lid hinge may be dirty and do not make contact: Clean the metal safety contacts in the lid hinge with a cotton swab and water.
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Yes, the iBlot Transfer Stacks are compatible with Li-COR detection.
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The Blotting Roller is a plastic roller attached to a stainless steel handle. It is used to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks and gel.
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The De-bubbling Roller is a stainless steel, aluminum roller designed to remove any air bubbles between the gel and blotting membrane during the assembly of the stacks when blotting E-PAGE gels. We recommend using the De-bubbling Roller only for E-PAGE gels. Other gel types may stretch and tear if pulled through the roller. The De-bubbling Roller is provided with the iBlot Dry Blotting System but not with the iBlot 2 Dry Blotting System.
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The iBlot E-PAGE Tab is a steel tab used during blotting of E-PAGE gels. It is attached to the iBlot Cathode Stack, Top and is used to pull the transfer stack assembly towards the blotting surface during the de-bubbling process of E-PAGE gels.
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The iBlot Filter Paper is used for blotting mini- or midi-gels. It is placed on top of the gel before placing the iBlot Cathode Stack (Top Stack) to protect the gel integrity during the blotting process. The iBlot Filter Paper is supplied in two sizes for efficient blotting of mini- and midi-gels. We do not recommend using the iBlot Filter Paper for blotting E-PAGE gels.
Note: Failure to use the iBlot Filter Paper during blotting of mini- or midi-gels may result in high currents exceeding the current limit leading to an error (Error2) during the run.
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A conventional stripping protocol using 0.1 M glycine, pH 2, works with polyclonal antibodies.
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Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from the gel to the membrane. We recommend using P3 program with a transfer time of 8-10 minutes for optimal transfer of these proteins.
To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE Invitrogen 3-8% Tris-acetate gels for electrophoresis. For the protocol, please see Transferring Large and Small Proteins Using the iBlot Dry Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/protein-expression/pdfs.par.87290.file.dat/iblot%20small%20and%20large%20protein%20app%20note.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The electrodes used in our iBlot Transfer Stacks are copper-coated nylon, and the amount of copper left in the electrodes after a transfer is rather small. There is 0.6 grams of copper per sheet before transfer, and even less after transfer. To maximize the recovery of the copper mesh electrodes, we have established an agreement with a recycling center in the United States. In order to prepare the copper electrodes for recycling, please follow the instructions listed here (https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.0358.file.dat/iBlot%20copper%20reycling%20instructions%20NA%20only.pdf).
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It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The iBlot Gel Transfer stacks should not be frozen as they contain a gel matrix that will be damaged by freezing.
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Yes, the Cat. No. for the iBlot lid latch is IB1003 and the Cat. No. for the iBlot Electrodes (1 pair) is IB1002. Here are the instructions to replace the electrodes (http://tools.thermofisher.com/content/sfs/manuals/iblot_electrode_replacement_man.pdf).
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The PVDF membrane is pre-activated and ready for use without any pretreatment with alcohol.
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We recommend storing the iBlot Transfer Stacks at room temperature.
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The following iBlot Transfer Stacks are available for use with the iBlot Gel Transfer Device and are also sold separately:
- iBlot Gel Transfer Stacks are used to transfer proteins from gels onto nitrocellulose or PVDF membranes (western blotting). Both nitrocellulose and PVDF transfer stacks are available in mini and regular formats.
- iBlot DNA Transfer Stacks are used to transfer DNA from gels onto nylon membranes (Southern blotting).
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The iBlot Dry Blotting System is compatible for use with Bolt Bis-Tris Plus, NuPAGE Bis-Tris, Tris-Acetate, Tris-Glycine, Tricine (in mini- and midi-gel formats), and E-PAGE gels.
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The iBlot Gel Transfer Device has been discontinued and we have launched the iBlot 2 Gel Transfer Device, that is an improved version. The iBlot Transfer Stacks, Western Detection Stacks and DNA Transfer Stacks are still available for purchase and are to be used exclusively with the original iBlot Gel Transfer Device, and are not compatible with the new iBlot 2 Gel Transfer Device (http://www.thermofisher.com/order/catalog/product/IB21001). Only iBlot 2 consumables are to be used with the iBlot 2 Gel Transfer Device.
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Yes, please take a look at the Western Blotting NativePAGE Invitrogen Bis-Tris Gels Using the iBlot 7-Minute Blotting System Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/en/filelibrary/pdf.par.18870.file.dat/native-with-iblot-app-note-v3.pdf).
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