Here are the easy step-by-step instructions on how to use the Glow-Writer Pen. Please read all steps below prior to use. The most effective time to annotate the blot is immediately before the addition of substrate.
Shake the pen vigorously for 30 – 45 seconds prior to each use.
On a paper towel, press down on the pen’s nib. Keep the nib pushed in until you see green ink saturate the nib. This may take 30 seconds to a minute.
Before attempting to write on a blot, ensure that the pen writes smoothly without having to push down on the nib.
NOTE: It is best to test the pen on a piece of lab tape or parafilm just before writing on the blot to ensure that the pen is writing smoothly.
Trace the visible ladder on the membrane. It may be necessary to wick excess fluid off the membrane using a tissue prior to annotation. Lightly touching the pen to the membrane should be enough to transfer ink to the membrane.
NOTE: Do not push down on the nib so hard that it enters the pen chamber while the pen is on the membrane. Doing so will flood the membrane with ink.
Substrate can be added immediately after writing on the membrane with the Glow-Writer pen.
After substrate incubation, expose the membrane to light for 30 – 60 seconds. Leaving the membrane on the bench top should be sufficient light exposure to “excite” the ink. If bands are not bright enough, closer exposure to a light source may increase band brightness. Ink glows for approximately 5 minutes.
Traditional Western blotting is a labor-intensive process that includes gel electrophoresis, protein transfer to a blotting membrane, incubation with primary and secondary antibodies, and chemiluminescent or fluorescent detection of target proteins. (View a typical Western blotting workflow.) Day-to-day reproducibility is poor, because small variations in lysate preparation, gel loading, electrophoresis, transfer, and detection are unavoidable sources of technical variability.