Protocol: Gel Electrophoresis & Protein Transfer by Electroblotting

* Detailed instructions for making gels in-house can be found in Sambrook et al.

  1. For standard denaturing and reducing conditions, mix sample 1:1 by volume with Laemmli 2x sample buffer.
  2. Heat to 100oC for 5 minutes or 70oC for 10 minutes.
  3. Load 20-50 µg sample per lane, along with suitable positive and negative controls.
  4. Run the gel and transfer to PVDF or nitrocellulose membrane according to the manufacturer’s instructions for the equipment and materials in use.
  5. Confirm protein transfer by staining for 1 minute with Ponceau S, followed by complete destain in distilled water.
  6. Mark position of molecular weight standards if they are unlabeled.

(For our in-house Western blotting, we use NuPAGE® Novex® precast 4-12% gradient gels run in Tris-Acetate buffer and transferred to nitrocellulose using the Xcell SureLockTM II blot module from Invitrogen, as per the manufacturer’s instructions.)



Western Blot Protocol: Cell Lysis, Mammalian Cells Western Blot Protocol: Immunodetection - Direct and Indirect