(Eliminates time consuming transfer of glass plates between separate casting and gel running components) Complete system allows casting of gels and running of gels, simultaneously, increasing efficiency.Electroblotting and PAGE gel electrophoresis in same universal gel tank.Water Bath, Shaking Water Bath, Bead BathĮNDURO™ VE20 Dual Casting System, 20x20cm (E2020-CU).Sometimes just making fresh buffer can solve a world of problems. Variables to address are methanol content, SDS content, salt concentration, and pH. Research the buffer that works best in your system and with your membranes. Depending on your membrane and proteins of interest, your buffer may be to blame. Is it still operational? Did you set it at the correct volts/amps? Check the coating on your anode for wear, tear, and scratches. If they are reversed, your proteins will be pushed away from the membrane and float away in the buffer. Solution: Black in back, red ahead! Make sure that your wires are attached to the correct electrode.There could be a problem with your set-up that is keeping you from success. If you are sure that the above culprits aren’t the issue, check the function of your transfer apparatus and power source or your buffers. Delicacy is the order of the day.Ĭommon Problem #6 – Transfer Apparatus or Buffer Component Issues. Although they are relatively hardy, the truth remains that we’re dealing with thin, gelatinous sheets.

Solution: Use a squeeze bottle with water to shoot liquid in between the gel and the plate to further loosen the gel before trying to remove it from the plate.Solution: Run a razor blade along the edges of your gel to separate it from the plate.Gels are notorious for sticking to the glass plates when removing them from the SDS-PAGE apparatus. However once again it is vital to stain your membrane with Ponceau S to visualize and mark the problem area. This doesn’t have to spoil your transfer, as the rest of the blot is probably fine. When removing a gel from between the glass plates, placing it in the transfer apparatus, and assembling the sandwich, it is very easy to tear the gel. One of the main reasons that we transfer proteins from gels to membranes before probing with antibody is that gels are so fragile. At each step in the process (filter paper – gel – membrane – filter paper) it is important to look for and remove any bubbles. One of the most important things to do while setting up your transfer is to be observant and mindful as each layer is added. This is similar to the beer-against-the-side-of-the-glass technique. Solution – Pour the transfer buffer slowly into your tank to keep bubbling at a minimum.Pouring transfer buffer into transfer apparatus too quickly.Solution – Mix fresh buffer without shaking – use a stir bar.Solution – Degas your transfer buffer.The agitation of mixing transfer buffer causes bubbling that can then get trapped. This allows me to use the rest of the blot without worry. Then when I use the blot later and the Ponceau S is washed away during blocking and incubation steps, I will still be able to see the compromised portion of the blot. Usually bubbling is minor, and if it is just a couple of bubbles like in the blot above, then I simply use a gel pen to circle them on the blot. Bubbles are the result of air being trapped between the membrane and the gel during transfer. A bubble appears as a white spot on your membrane in the middle of the field of red Ponceau S stain.

Horizontal compression on sds page gel