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.
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