How to automate your western blot processing using the Invitrogen iBind Western Systems

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After western blot transfer, the membrane is ready to be probed for your protein, or proteins, of interest. Traditionally, this has been a manual process that involves incubating the blot in a series of antibody and wash solutions in a tray over several hours. Invitrogen iBind Western Systems are non-powered, automated western-processing devices that allow all solutions to be prepared and loaded in the device at the start of the procedure, from which point all steps proceed automatically and uninterrupted by sequential lateral flow technology. In this video, you’ll learn how to use the iBind western systems. Two iBind Western Systems are available: the original iBind Western Device, which accommodates one mini blot at a time, and the iBind Flex Western Device, which accommodates two mini blots, one wide-format midi blot, or up to six vertically cut strips at a time. The iBind Flex uses interchangeable well inserts to accommodate the blot format. To access the different inserts, open the spring loaded storage drawer. To change the inserts, push out from inside of the top lid and drop the new insert into place. Note that it can only be installed in one orientation. Start by Preparing your 1X iBind solution, and your primary and secondary antibody solutions according to the quick reference card. Next place your blot or blots in 10 mL of 1X iBind solution after the transfer step. Remove an iBind card from the pouch, taking care to handle it by the bottom thicker portion only. Place the card in the device, making sure it is aligned against the backstop. Do not use cards that have been bent or damaged. Wet the card evenly with 10 milliliters of 1X iBind solution. Once wet, alignment guides will show through the card. Use them to ensure proper alignment of mini blots or vertically cut strips with their respective wells. To process a midi blot, first make sure the midi well insert is installed. Then add an additional 2 milliliters of 1X iBind solution to the card, allowing it to pool. Place your blot protein-side-down across the membrane region indicated on the device, with low molecular weight bands closest to the stack. To process up to two mini blots, first make sure the mini well insert is installed, then add an additional 1 milliliter of 1X iBind Flex solution per blot to the card, allowing it to pool. For mini-sized blots, use the center alignment guide to place your blots on either side. For up to six vertically cut strip blots, start by installing the multi-strip well insert. Add an additional 2 milliliters of 1x ibind flex solution to the card, allowing it to pool. Use the alignment guides to place your blots in the white spaces between the black lines. Using the roller, firmly roll the membrane to remove any air bubbles and to ensure good contact between the membrane and the card. Close the lid and open the liquid door to expose the wells. Referencing the volumes in the quick reference guide- add your primary antibody to each well of row 1. Add 1X iBind solution to each well of row 2, your secondary antibody to each well of Row 3, and 1X iBind solution to each well of row 4. For mini and multi-strip inserts, each column can be loaded with a different antibody pair. Close the lid and use a dry-erase marker to indicate that the device is in use and note the start time of the experiment. The blots can be removed after two and a half hours or can be left overnight. When you return, open the liquid door to ensure that all the liquids has moved into the card. Any remaining liquid indicates that the experiment has not yet completed. Once completed, rinse your blot in water and dispose of the used iBind card. Proceed to your chemiluminescent, fluorescent, or colorimetric western detection protocol. Get more information about Invitrogen iBind western systems at thermofisher.com/iBind

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