The transfer sandwich must apply equal and firm pressure across the entire blot. Most of them can be solved by tightly packing the transfer sandwich. Tightly Packing the Transfer Sandwich can help avoid Vertical Variation / Horizontal Waves / Smudged Banding.Īll three of these issues are very common. You can minimize bubbling be degassing your buffers, avoiding bubbling creation by pouring the buffer slowly when filling the tank, and carefully assembling your transfer sandwich by prewetting your sandwich pads and using a roller at each step to press out any trapped bubbles in the sandwich. This will allow you to visualize the location of the bubbles once the Ponceau S is washed away in the incubation steps. A couple of bubbles and a small smudge can be easily avoided thanks to the stain’s visualization.Īir bubbles trapped between the gel and the membrane can leave white circles on your blot.Īfter staining with Ponceau S, use a gel pen to outline these bubbles on your blot. The rat brain blot below illustrates a beautiful transfer for mid-range MWs. Ponceau S staining is a wonderful way of illustrating the transfer efficiency across the whole blot, but also for highlighting any issues with small sections that could be problematic for that specific area, but that don’t negatively impact your entire blot. Protein Visualization is Key to a Successful Western Blot Read my full-length piece in our website’s Education section.
#WESTERN BLOT SANDWICH ORDER HOW TO#
Here's how to avoid them - or at least how to see they are there. There are several blot blunders that can mess with your transfer.
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