Multiplexing 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 and provide results for a possible maximum of 48 targets on a single membrane — 24 per channel with ® Imagers 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.
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:
Install Image Studio on your computer before connecting the C-DiGit Blot Scanner. Incubate using room temperature substrate.
Wrap your blot so it stays wet during the scan.
Remember to place your blot protein side down.
If sensitivity is an issue, use
WesternSure™ PREMIUM or SuperSignal ® West Femto Chemiluminescent Substrate. Start with high sensitivity scan for your first scan and then work from there.
Image on the
C-DiGit Scanner first and then exposure your film.
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.
The 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.
The 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.