iBind™ Cards, 40 cards - FAQs

To strip and reprobe your blot processed using the iBind Western Device or iBind Flex Western Device, first strip the blot outside the system using your preferred protocol. After the stripping protocol, wash the blot in distilled water for 5 min. Reblocking is unnecessary as the iBind Solution will perform this step during reprobing. Finally, reprobe with new antibodies using the iBind Western Device or iBind Flex Western Device, starting with the membrane preparation step.

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Yes, for each midi blot, mini blot, or vertically cut strip, prepare appropriate primary antibodies together in a single tube of iBind FD/ iBind Flex FD Solution, Prepare appropriate secondary antibodies together in a different tube of iBind FD/ iBind Flex FD Solution. Do not mix primary and secondary antibodies prior to loading in the device. All primary and secondary antibodies should be diluted according to the recommendations outlined in the user manual or quick reference card. When detecting several targets of the same species, the same concentration of secondary antibody can be used as when detecting one target. There is no need to increase the concentration for each additional target. Load multiplexed antibodies into the device as normal.
Caution: Consider cross-reactivity of secondary antibodies when multiplexing (e.g., rabbit anti-goat IgG and goat anti-mouse IgG are likely to cross-react).

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Yes, both the iBind Western Device and iBind Flex Western Device work well with Alexa Fluor Plus conjugated secondary antibodies.

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When firmly rolling, the iBind/iBind Flex Card will slightly dip in the membrane area, causing the top and bottom of the card to lift. See the iBind/iBind Flex user manual for additional details.

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Besides being ~1.7 times as wide, the iBind Flex Card also includes alignment lines printed on the back that appear when solution is added to the card to help in proper horizontal placement of multiple mini or vertically cut strip blots.

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The formulations are identical for both the standard and Fluorescent Detection (FD) solutions. The only difference is in the amount supplied, so the iBind Flex Solution Kits are sufficient for 10 midi, 20 mini, or 60 vertically cut strip blots.

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Yes, to run a single mini blot or up to three vertically cut strip blots. However, when using the Mini or Multi-Strip Inserts, wells not in contact with the card should remain empty.

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Please use the protocol mentioned on Page 13 of the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ibind_man.pdf) when using the iBind Western System in conjunction with the LI-COR Odyssey Imaging System. We do not recommend using the LI-COR solution.

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If the PVDF membrane has dried up, it is important to pre-activate it with 100% methanol and then rinse in distilled water before immersing it in iBind Solution/iBind Fluorescent Detection (FD) Solution. It is not necessary to activate a PVDF membrane that has just come out of the transfer and moved into iBind Solution/iBind Fluorescent Detection (FD) Solution.

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Rinse the iBind Western Device under running water after each use and allow the device to dry before additional usage. No use of detergent is recommended as any residual detergent may interfere with results. To maximize the life of the springs in the iBind Western Device, store the device with latch unlocked, and the lid open as shown on Page 23 of the manual (https://tools.thermofisher.com/content/sfs/manuals/ibind_man.pdf).

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It is sufficient to block the membrane in iBind Solution/ iBind Fluorescent Detection (FD) Solution for the time prior to making antibody dilutions. It is not necessary to block the membrane overnight in the blocking solution. The membrane will be subjected to additional blocking in the iBind Solution/ iBind Fluorescent Detection (FD) Solution before primary antibody reaches the membrane.

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Yes, membranes that are smaller than 9 cm x 9 cm can be used with the iBind Western device. It is recommended to place the strips or smaller blots centrally. When using larger membranes, care must be taken to ensure that no part of the membrane is in contact with the stack on the iBind card.

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We do not recommend using your own solutions. The iBind Solution and iBind Fluorescent Detection (FD) Solution have specific viscosity and are optimized for the sequential lateral flow that is the principle of the iBind Western device. We cannot guarantee the performance with any other solutions.

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The original iBind Western System (Cat. No. SLF1000) consists of:
- iBind Western Device
- iBind Blotting Roller
- iBind Window Cover

You would need to purchase the following items separately:
- iBind Cards (Cat. No. SLF1010)
- iBind Solution Kit (Cat. No. SLF1020) for preparing blocking, dilution, and washing buffers for chemiluminescent or chromogenic detection
- iBind Fluorescent Detection Solution Kit (Cat. No. SLF1019) for preparing blocking, dilution, and washing buffers for fluorescent detection
- Invitrogen AP Mouse Chemiluminescent Detection Kit (Cat. No. SLF1021) or Invitrogen AP Rabbit Chemiluminescent Detection Kit (Cat. No. SLF1022)

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iBind Western Systems are benchtop devices that automate immunodetection steps. These devices use no external power source, and rely on mechanical pressure to generate sequential lateral flow (SLF) of immunodetection reagents to perform hands-free blocking, antibody binding, and washes for western detection workflows.

Two iBind Western Systems are available:
i) containing the original iBind Western Device, which accommodates one mini blot at a time
ii) containing the iBind Flex Western Device, which accommodates one or two mini blots, one midi blot, or up to six vertically cut membrane strips (for separate antibodies) at a time.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.