Note: Ensure that both plates are flush on a level surface and that the label on the spacer plate is oriented correctly. Leaking may occur if the plates are misaligned or oriented incorrectly.
Note: The gray casting stand gaskets must be CLEAN and DRY. The casting stand gaskets are made of a special thermoplastic material that swells when soaked in water, so we recommend that you DO NOT soak the gaskets for prolonged periods prior to casting. If the gaskets do get accidentally soaked and display swelling and/or deformation, just allow them to air dry and they will regain their original shape, size, and performance.
Note: When running 2 gels only, use the electrode assembly (the one with the banana plugs), NOT the companion running module (the one without the banana plugs). When running 4 gels, both the Electrode Assembly and the Companion Running Module must be used, for a total of 4 gels (2 gels per assembly).
Note: It is critical that gel cassettes are placed into the clamping frame with the short plate facing inward. Also, the clamping frame requires 2 gels to create a functioning assembly, If an odd number of gels (1 or 3) is being run, you must use the buffer dam.
Note: Do not attempt to lock the green arms of the clamping frame, without first ensuring that the gel cassettes are perfectly aligned and stabilized against the notches on the green gaskets of the module. To prevent the gels from shifting during the locking step, firmly and evenly grip them in place against the core of the module with one hand.
CAUTION: When running 1 or 2 gels only, DO NOT place the Companion Running Module in the tank. Doing so will cause excessive heat generation and prevent electrophoretic separation.
Load 10 μL (for immunoblot purposes or 5 μL for staining purposes) of the Precision Plus Protein Standard Dual Color on lane 1.
Load 20-40 μL of each of your protein samples per lane, depending of the gel thickness.
Note: You may use a sample loading guide to locate the sample wells. Place the pipette tip into the slots of the guide and fill the corresponding wells.
CRITICALLoad samples slowly to allow them to settle evenly on the bottom of the well. Be careful not to puncture the bottom of the well with the syringe needle or pipette.
If your purpose at the end is to have a beautiful image you have to run the gels at 100V; use 200V when time is important.
Note: Always pour off the buffer before opening the arms of the assembly, to avoid spilling the buffer.
a) This will help you to always visualize the gel in the proper orientation (in other words in the order that you loaded your samples).
b) Lane 1 will always be protein standard.
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SDS-PAGE (Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis) is a method for separating proteins by molecular weight. Recipes for buffers can be found on the western blot recipes sheet.
Protocol Authors: Osvaldo D. Rivera, PhD & Luis Vazquez Quiñones, PhD