Western blotting is a frequently used application for the qualitative & quantitative analysis of protein expression levels in a variety of biological samples. The typical Western Blot workflow includes protein separation, transfer to an immunoblot, protein detection and finally Western Blot qualification & quantitation.
Need help optimizing your Western Blot experiments? Check out our tools for Western Blot optimization, full of guides, protocols, webinar series and more!
After you have prepared your sample, you need to load your sample on an electrophoresis gel to separate your protein of interest from the other proteins.
There is a variety of electrophoresis gels and buffers on the market: the choice of gel and buffer depends on your application and the conditions you have to meet for your protein, like size, structure, charge or any of these characteristics combined.
To be able to detect your protein of interest you need to immobilize it onto a membrane. To make sure your protein is transferred completely and efficiently onto the membrane, there are various Western Blot systems, membranes, blotting buffers and more to choose from.
Once you have transferred your proteins succesfully onto a membrane, protein detection is the next step. The most well-known strategy for protein detection is chemiluminescent imaging using HRP-labeled antibodies. Alternatively, you can choose for fluorescent imaging by using fluorescently labeled antibodies. This technique is more advantageous due to the ability to detect multiple targets on the same blot and at the same time, without the need for stripping or reprobing, no worries about incubation times or film exposures, and saving you time and money on consumables.
Analysis & Quantitation
Finally, you need to image and analyze your blots in order to qualitate & quantitate your results. After all, you're looking for differences in protein levels, right?
To quantitate your results the most common used method is chemiluminescent detection on film. However, if you're looking for more accurate and reproducible results, digital imaging or near-infrared technology is the way to go, securing you with more reproducible and quantifiable data for publication and funding.