Running Invitrogen NuPAGE gels

Sample preparation

  1. Prepare the sample:
    Reagent
    Sample
    LDS sample buffer (4x)
    Reducing agent (1x)
    Deionized water

    Reduced sample
    x µl
    2.5 µl
    1 µl
    to 10 µl

    		 Non-reduced Sample
    x µl
    2.5 µl

    to 10 µl
  1. Heat the sample at 70°C for 10 minutes.

Preparing running buffer

  1. Prepare 1L of 1X running buffer from the NuPAGE SDS 20x stock.
  2. Just prior to electrophoresis, add 500 µl of NuPAGE Antioxidant to 200 ml of 1X running buffer for use in the upper buffer chamber. For non-reducing gels, leave out the antioxidant.

Electrophoresis

  1. Rinse the gel cassette with deionized water, peel of the tape and remove the comb.
  2. Rinse the wells 3 times with 1X running buffer.
  3. Put the gels in the XCell SureLock apparatus with the well sides facing inwards, and lock them into place with the tension wedge. Use the plastic buffer dam if you use one gel.
  4. Fill the upper (inner) buffer chamber with running buffer.
  5. Load the gel.
  6. Fill the lower buffer with 600 ml of 1X running buffer.
  7. Run the gels according to the following table:

    Gel Type Voltage Expected Current Run Time
    Novex Bis-Tris Gels
    with MES SDS buffer    		 200 V

    Start: 110-125 mA/gel
    End: 70-80 mA/gel

    		 35 min.
    Novex Bis-Tris Gels
    with MOPS SDS buffer    		 200V Start: 110-115 mA/gel
    End: 60-70 mA/gel    		 50 min.
    Novex Tris-Acetate Gels 150V Start: 40-55 mA/gel
    End: 25-40 mA/gel    		 1 hour
    Novex Tris-Acetate Native Gels 150V Start: 18 mA/gel
    End: 7 mA/gel     		~2 hours