How to perform a western wet tank transfer using the Invitrogen Mini Blot Module

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Once the proteins have been separated in the gel, they now can be transferred to a solid support membrane to start the western blotting workflow. In this video, you’ll learn how to perform a western transfer using the Mini Blot module in a Mini Gel Tank. Make sure the cassette clamps are removed before you set up your transfers. One gel is transferred per module, and two modules fit in one tank. Included with the Mini Blot Module kit is a roller, sponge pads, tweezers, and a quick reference card. Approximately 250 milliliters of Transfer Buffer is required for each transfer. Prepare Transfer Buffer by adding 25 milliliters of 20X Bolt Transfer Buffer, 50 milliliters of methanol, and 500 microliter of Bolt Antioxidant, and bring the total volume up to 500 milliliters with deionized water. Soak two sponge pads in transfer Buffer and Squeeze the pads to remove air bubbles. Open the gel cassette using a gel knife and carefully remove the wells and the foot of the gel, so that the entire gel is of equal thickness. Place a shallow dish on the bench and pour 1X transfer buffer to dip the filter papers into during sandwich assembly. Assemble the western transfer sandwich by placing the cathode or negative core on a flat surface. Pour 5 milliliters of transfer buffer into the core, then place a pre-soaked sponge pad on the cathode core followed by a pre-wetted filter paper. Next, use the blot roller to squeeze out the air bubbles from between each layer. Place the gel on the stack, again using the roller to remove air bubbles. Using tweezers, place the membrane on top of the gel and roll once more. Complete the stack by layering another pre-wetted filter paper on top of the membrane, removing bubbles, and placing pre-wetted sponge pads on top of the filter paper. Complete the assembly by placing the anode core on top of the sandwich and pressing the two module halves together. Insert the blot module into the Mini Gel Tank with the cathode core facing the front. The blot module should be seated with the electrodes contacting the electrode bar. Extra transfer buffer can be added to the module core to submerge the sandwich, but don’t fill it above the gasket. Add transfer Buffer or deionized water—about 225 milliliters to the chamber, just below the electrode bar. Make sure the power supply is off. Place the cover on the tank and plug the power leads into the power supply. Turn the power supply on to begin the transfer. For Nitrocellulose membranes, transfer at a constant 10 volts for 60 minutes. For PVDF membranes, transfer at 20 volts for 60 minutes. After the transfer is complete, turn off the power supply, disconnect the cords, open the lid by placing your thumb on the tank handles pulling the lid up with your fingers. Remove the module, pour out the buffer, and disassemble the module. Remove the membrane using tweezers. You are now ready to probe your membrane for western analysis. Find out more information about western blotting with the Mini Blot Module at thermofisher.com/westerntransfer

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