iBind™ Flex Western Starter Kit, fluorescent detection - FAQs

Yes. When using a conjugated primary antibody and no secondary antibody, add the diluted primary antibody to well 1 and replace diluted secondary antibody (well 3) with 1X iBind Flex/iBind Flex FD Solution. Alternatively, for faster time to results, add the diluted primary antibody to well 1, leave wells 2 and 3 empty, and add 1X iBind Flex/ iBind Flex FD Solution to well 4. This procedure will save time (~60 min) but may result in higher background.

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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.

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