How to perform a western blot semi-dry transfer using the Invitrogen Power Blotter with Pre-cut Membranes and Filters

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In this video, you’ll learn how to transfer proteins from polyacrylamide gels to membranes using the Invitrogen Power Blotter with our pre-cut membranes and filters, and Power Blotter 1-Step Transfer Buffer. The Power Blotter 1-Step Transfer Buffer is an optimized, high-ionic strength buffer that facilitates rapid transfer conditions when combined with the high-current conditions of the Power Blotter System. Filter papers soaked in the 1-Step Transfer Buffer act as an ion reservoir. The system can also be used for standard semi-dry transfer protocols that use traditional buffers. In the Power Blotter System, the cathode and anode are plates that are fixed to the top and bottom of the cassette frame, and the transfer stack is placed in between them during the transfer process. The Power Blotter system has two different cassette sizes: a standard size for transferring one midi gel or up to 2 mini gels, and the XL cassette for transferring up to 2 midi gels or up to 4 mini gels at one time. To begin the transfer process, flip the power switch at the rear of the device to turn on the system. The Power Blotter 1-Step Transfer Buffer comes in a 5X concentration. Before using, dilute to a 1X concentration using deionized water. Equilibrate membrane and filter papers in the diluted Power Blotter 1-Step Transfer Buffer for a minimum of 5 minutes. Use enough buffer to completely cover the filter papers and membrane. Four pieces of filter paper are required for each membrane. When using a PVDF membrane, be sure to wet the membrane with methanol or ethanol before soaking in 1X Power Blotter 1-Step Transfer Buffer. Open the Power Blotter Cassette by pressing the grey button on the lid. Remove 2 pieces of filter paper from the buffer and place them in the center of the bottom part of the cassette. Place the membrane on top of the filter papers and remove any trapped air bubbles using the blotting roller. Immerse the pre-run gel in deionized water for 5–15 seconds. Place the pre-run gel on the transfer membrane. Trim the gel carefully so that no parts of the gel overhang the sides of the stack. Use the roller to remove any air bubbles from between the gel and the membrane. Place the other two pre-wetted filter papers on top of the pre-run gel and use the blotting roller to remove any air bubbles from between the gel and filter papers. If transferring more than one gel, ensure there is a 1 centimeter of space around all stack edges. Place the cathode lid on top of the anode and gently press down on the top of the cassette to lock it into place. Slide the assembled cassette into the Power Blotter Station. The Power Blotter system has preprogrammed transfer methods that are based on the number of gels being transferred and the molecular weight range of your proteins of interest. Custom methods can also be created. After selecting or creating a method, simply press Start to begin the transfer. The system signals the end of the transfer with repeated beeping sounds and a message on the display. Upon transfer completion, pull the cassette out from the base and open the cassette. Carefully remove and discard the two top filter papers and the gel. Use forceps to remove the transfer membrane. Discard the two bottom filter papers. Now your membrane is ready to be blocked and probed for your specific proteins. When performing several consecutive transfers, drain the cassette by tilting the cassette at the corner and wipe down the anode surface with a damp cloth or tissue paper to remove any excess liquid. The Power Blotter system is ready for another run with no cooling period required unless the system has been running continuously for 2 hours. After extended use, cool the cassette for 30 minutes. Get more information about the Invitrogen Power Blotter at thermofisher.com/power blotter

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