To dry, or not to dry: that is the question. After a long day preparing your fluorescent Western blot, it is tempting to rush to imaging so that you can see the fruits of your labor. There’s nothing wrong with this work-flow but you will likely not generate the best possible images. If you are running a qualitative experiment or have a highly abundant protein, imaging the membrane wet is fine. However, if you are like most researchers, you have turned to fluorescent Western blotting for quantitative analysis of your low expressed target.
Wet membranes are not ideal for imaging because they slowly dry non-uniformly, causing inconsistencies in your data. Since wet membranes auto-fluoresce differently than dry membranes, special care needs to be taken to prevent membranes from drying out during the process of imaging. This means adding buffer to the imaging area and making a big mess or placing your blot in plastic wrap to prevent drying. If you have ever imaged blots wet, you know that preventing drying during long exposures is a hassle. One of the great advantage of working with fluorescent blots is supposed to be imaging at your convenience. However, if you have to fuss with wet blots and store them in trays filled with buffer, it is not very convenient at all! Besides the extra hassle, it is not uncommon to see high levels of general background when wet membranes undergo long exposure times. High general background negatively impacts sensitivity, making detection of low abundance targets even more challenging.
Imaging membranes wet is clearly an inconvenience and far from ideal, but that doesn’t mean you have to wait hours for your membrane to dry out either. A quick way to dry out your membrane after fluorescent Western blotting is to dip it in methanol. All it takes is a quick 15 second dip in 100% methanol for PVDF membranes. Nitrocellulose membranes are not as resistant to chemicals so use 20% methanol instead. The alcohol will displace the water from the pores of your membrane to accelerate drying and allow you to image your blot sooner. This quick tip will get you to a beautiful hassle free blot in no time!
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