Easily Annotate Protein Molecular Weight Ladders on Chemiluminescent Western Blots

Glow-Writer Pen for Chemiluminescent WesternsThe Glow-Writer pen (LI-COR P/N 926-90000) is a phosphorescent marker used to annotate visible protein molecular weight ladders for chemiluminescent Western blot detection. The procedure described below has been optimized for detection using the C-DiGit® Blot Scanner or the Odyssey® Fc Imaging System. The Glow-Writer pen may be suitable for use with film or other imaging systems; however, further optimization may be necessary.

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.

  1. Shake the pen vigorously for 30 – 45 seconds prior to each use.
  2. 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.
  3. 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.
  4. 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.
  5. Substrate can be added immediately after writing on the membrane with the Glow-Writer pen.
  6. 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.
  7. Re-expose to light if blots are imaged again.

Get a Glow-Writer Pen today so that you, too, can easily annotate protein molecular weight ladders and ensure that you can detect this important size on film or chemiluminescent Western blot imaging systems.

Multiplex Western Blotting System Turbo-Charges Western Blot Results Output

Example of Multiplexed Western Blot using the MPX Blotting SystemMultiplexing is a powerful tool that allows you to get more out of your Western blots. Multiplex detection becomes possible when you utilize the MPX™ (Multiplex) Blotting System and LI-COR IRDye® near-infrared fluorescent dye-labeled secondary antibodies.

Multiplex Westerns can be imaged on any of the Odyssey® Imagers and provide results for a possible maximum of 48 targets on a single membrane — 24 per channel with two-color detection — and the option for quantitative analysis, saving you time and reagents! The MPX Blotting System can be used if you need to optimize:

Watch this 4 minute video on how easy it is to get the most out of multiplexing with the MPX Blotting System. You can also download the handy MPX Blotter User Guide.

5 Technical Tips for Chemiluminescent Western Blotting Success with the C-DiGit® Scanner

In this short video, Jessica talks about 5 tips to help ensure that imaging chemiluminescent Western blots on the C-DiGit Blot Scanner is a success – the first time and always!

Here’s a recap of the tips of those five technical tips:

  1. Install Image Studio on your computer before connecting the C-DiGit Blot Scanner.
  2. Incubate using room temperature substrate.
  3. Wrap your blot so it stays wet during the scan.
    1. Remember to place your blot protein side down.
    2. If sensitivity is an issue, use WesternSure™ PREMIUM or SuperSignal® West Femto Chemiluminescent Substrate.
  4. Start with high sensitivity scan for your first scan and then work from there.
  5. Image on the C-DiGit Scanner first and then exposure your film.

C-DiGit Blot ScannerHappy Blotting!

Rethinking the Traditional Western Blot

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.

Snapshot of In-Cell Western Assay MethodThe In-Cell Western™ (ICW) Assay, a quantitative immunofluorescent method, is an alternative to traditional Western blots that increases both reproducibility and sample throughput. (View a typical ICW workflow.)

We recently hosted a webinar called “Rethinking the Traditional Western Blot”, during which John Lyssand, PhD, from LI-COR Biosciences, discussed the In-Cell Western Assay and its use in neuroscience research, in this case, Alzheimer’s Disease. The In-Cell Western Assay enables screening and analysis of many more samples in each experiment, eliminates error-prone protocol steps, and delivers higher reproducibility for biological and technical replicates.

ICW Use: Tau Protein Accumulation and InhibitionThe data presented demonstrated how ICW assays were used in Alzheimer’s Disease research to screen HSP90 inhibitors for their effectiveness in reducing tau activity levels. Dr Lyssand discussed how and why the In-Cell Western Assay is superior to traditional methods for screening of cell samples.

If you didn’t have a chance to join us in September for “Rethinking the Traditional Western blot”, you can view this webinar online and on-demand. Check out the information on In-Cell Western assays on our website. You can also read Professor Dickey’s white paper outlining how he and his group used In-Cell Western Assays to study Alzhemier’s Disease.