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        One-Dimensional SDS and Non-Denaturing Gel Electrophoresis of Proteins with Novex® Tris-Glycine Pre-Cast Gels

        互联网

        1069

        实验步骤

         

        1.        Protocol using XCell SureLock™ Mini-Cell

        1)        Remove the Novex® Pre-Cast Gel from the pouch.

        2)        Rinse the gel cassette with deionized water. Peel off the tape from the bottom of the cassette.

        3)        In one smooth motion, gently pull the comb out of the cassette.

        4)        Rinse the sample wells with the appropriate 1X SDS Running Buffer. Invert the gel and shake the gel to remove the buffer. Repeat two more times.

        5)        Orient the two gels in the Mini-Cell such that the notched “well” side of the cassette faces inwards toward the Buffer Core. Seat the gels on the bottom of the Mini-Cell and lock into place with the Gel Tension Wedge. Refer to the XCell SureLock™ Mini-Cell manual (IM-9003) for detailed instructions. Note: If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette.

        6)        Fill the Upper Buffer Chamber with a small amount of the running buffer to check for tightness of seal. If you detect a leak from Upper to the Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.

        7)        Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer. The buffer level must exceed the level of the wells.

        8)        Load an appropriate volume of sample at the desired protein concentration onto the gel (see Recommended loading volumes).

        9)        Load appropriate protein molecular weight markers.

        10)    Fill the Lower Buffer Chamber with 600 ml of the appropriate 1X running buffer.

        11)    Place the XCell SureLock™ Mini-Cell lid on the Buffer Core. With the power on the power supply turned off, connect the electrode cords to the power supply [red to ( ) jack, black to (-) jack].

        2.        Removing the Gel after Electrophoresis

        1)        After electrophoresis is complete, shut off the power, disconnect electrodes, and remove gel(s) from the XCell SureLock™ Mini-Cell.

        2)        Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the cassette’s two plates. The notched (“well”) side of the cassette should face up.

        3)        Push down gently on the knife handle to separate the plates. Repeat on each side of the cassette until the plates are completely separated. Caution: Use caution while inserting the gel knife between the two plates to avoid excessive pressure towards the gel.

        4)        Carefully remove and discard the top plate, allowing the gel to remain on the bottom (slotted) plate.

        5)        If blotting, proceed without removing the gel from the bottom plate.

        6)        If staining, remove the gel from the plate by one of the methods:

        7)        Use the sharp edge of the gel knife to remove the bottom lip of the gel. The gel knife should be at a 90° angle, perpendicular to the gel and the slotted half of the cassette. Push down on the knife, and then repeat the motion across the gel to cut off the entire lip. Hold the plate and gel over a container with the gel facing downward and use the knife to carefully loosen one lower corner of the gel and allow the gel to peel away from the plate.

        8)        Hold the plate and gel over a container with the gel facing downward. Gently push the gel knife through the slot in the cassette, until the gel peels away from the plate. Cut the lip off of the gel after fixing, staining, but before drying.

        9)        Fix and stain the gel as described.

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