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this step-by-step hands-on demonstration of western blotting is designed for those of you new to the technique making it easy for you to get started to ensure good results here are three things to think about before you start one that the sample is of good quality two that the electrophoresis gel is loaded optimally third that you select the correct electrophoresis conditions and wet or semi-dry transfer method ing to protein size and characteristics first prepare the gel for blotting by trimming off excess gel one corner of the gel can be cut off to help with orientation then equilibrate and transfer buffer for 10 minutes to maintain gel shape as with all pvdf membranes the cytiva high bond p blotting membrane must be pre-wetted in methanol rinsed in distilled water for 5 minutes then equilibrate it in transfer buffer for 10 minutes always use a blotting membrane of the same size and shape as the gel and the correct size for the frame build the sandwich carefully using two papers pre-