What types of membrane work best with SuperSignal West Pico PLUS Chemiluminescent Substrate?
Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Dura Chemiluminescent Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate. Please also see our guide for choosing western blot membranes (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html).
What types of membrane work best with SuperSignal West Femto Maximum Sensitivity Substrate?
Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Femto Maximum Sensitivity Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate. Please also see our guide for choosing western blot membranes (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html).
What types of membrane work best with SuperSignal West Dura Extended Duration Substrate?
Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Dura Extended Duration Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate. Please also see our guide for choosing western blot membranes (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html).
Following the release of the iBlot 2 Gel Transfer Device, are consumables for the original iBlot Gel Transfer Device still available?
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.
Is the iBlot 2 Gel Transfer Device compatible with original iBlot Transfer Stacks?
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.
How can I get better transfer of high-molecular weight proteins?
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)
Occasionally my western blots have high background. What do you recommend?
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.
Is it possible to substitute the membrane from the iBlot Transfer Stack with my specialized membrane?
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.
What is the shelf life of nitrocellulose and PVDF iBlot Transfer Stacks?
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.
Can iBlot Transfer Stacks be used more than once?
No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.
Do the PVDF iBlot Transfer Stacks (0.2 µm, non-autofluorescing) require activation prior to use?
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).
Sometimes there is green discoloration on the blot around the gel when using the iBlot Dry Blotting System. What is this, and does it affect the results?
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.
How can we be more environmentally responsible in regards to the packaging for the iBlot Dry Blotting System?
The plastic in the iBlot Dry Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.
I want to conduct western transfer with mini gels (8 x 8 cm), but I do not have iBlot Transfer Stacks (Mini). Can I use iBlot Transfer Stacks (Regular) to transfer my mini gels?
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.
Is there a limitation on the thickness of gels that can be used with the iBlot Dry Blotting System?
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.
What types of membranes are available in the iBlot Transfer Stacks?
iBlot Transfer Stacks are available with either an integrated 0.2 µm nitrocellulose membrane or a 0.2 µm PVDF membrane. Both types are available in two sizes: a mini size which can accommodate one mini gel (8 x 8 cm), or regular size which can accommodate one E-PAGE gel, one midi gel (8 x 13 cm), or two mini gels (8 x 8 cm).
The catalog numbers are as follows:
IB3010-01 iBlot Gel Transfer Stacks, Regular (10 Blots)
IB3010-02 iBlot Gel Transfer Stacks, Mini (10 Blots)
IB4010-01 iBlot Transfer Stack PVDF, Regular (10 Blots)
IB4010-02 iBlot Transfer Stack PVDF, Mini (10 Blots)
If you would like to blot proteins onto your own membrane rather than the supplied nitrocellulose or PVDF, you can replace the integrated membrane with your desired membrane as described below:
1) Wet the desired blotting membrane in deionized water. (For PVDF membranes, first wet the PVDF membrane in 100% methanol and then rinse in deionized water).
2) Carefully remove the nitrocellulose membrane from the bottom stack using forceps.
3) Place the wetted blotting membrane on the bottom transfer stack. (Membrane should be completely wet, but not dripping; make sure there is not too much excess water.)
4) Be sure to align the membrane flush to the bottom stack gel, and remove any air bubbles using the blotting roller. Proceed with the standard iBlot transfer protocol.
How can I get better transfer of low molecular weight proteins with the iBlot Dry Blotting System?
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).
With the iBlot Dry Blotting System, the iBlot Anode Stack, Bottom transfer gel melts to a viscous blue solution. How can I prevent this from happening again?
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.
I use the iBlot Dry Blotting System and see some corrosion of the iBlot Cathode Stack, Top. What is causing this?
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.
Are the iBlot Transfer Stacks compatible with Li-COR detection?
Yes, the iBlot Transfer Stacks are compatible with Li-COR detection.
What is the Blotting Roller that is provided with the iBlot Dry Blotting System?
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.
What is the De-bubbling Roller?
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.
What is the iBlot E-PAGE tab?
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.
What is the purpose of the iBlot Filter Paper?
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.
Is there a stripping protocol for the iBlot Dry Blotting System?
A conventional stripping protocol using 0.1 M glycine, pH 2, works with polyclonal antibodies.
How can I get better transfer of high-molecular weight proteins with the iBlot Dry Blotting System?
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).
Are the copper electrodes in the transfer stacks of the iBlot Dry Blotting System recyclable?
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).
I want to perform western transfer with mini gels (8 x 8 cm), but I do not have iBlot Transfer Stacks (Mini). Can I use iBlot Transfer Stacks (Regular) to transfer my mini gels?
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.
Is it okay to freeze the iBlot Gel Transfer Stacks?
The iBlot Gel Transfer stacks should not be frozen as they contain a gel matrix that will be damaged by freezing.
What is the recommended storage condition for the iBlot Transfer Stacks?
We recommend storing the iBlot Transfer Stacks at room temperature.
What are the different kinds of iBlot Transfer Stacks that you offer and are they available separately?
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).
