Western blotting is the method of isolating specific amino acid sequences from a material. Target proteins are tagged with specifically chosen primary and secondary antibodies, allowing you to see the particular protein of interest. Like any other scientific procedure, Western blotting systems must be performed accurately and comprehensively at each stage for excellent results.
To optimize western blotting, numerous steps might be taken. We know what works best because we have years of expertise. Jade Scientific, Inc. aims to offer help to save you time and make the procedure more straightforward and precise while maximizing your results. By maximizing the signal-to-noise ratio and limiting the appearance of non-specific bands, Western blot aims to produce the most precise results possible.
SDS-Page
We could go back to sample preparation, but let's start with sample separation for the sake of space. Long before handling your membrane, your Western blotting experiments begin. With this, you get what you put in; if you have a nice SDS-PAGE gel, your membrane will be excellent, and everything will stay intact.
Pick the Correct Membrane
Your membrane selection can significantly impact how well your Western blotting systems test turns out. The two most common membrane types are PVDF and nitrocellulose. However, several variations of each are now available on the consumables market, such as membranes designed for low-molecular-weight proteins or immunodetection based on fluorescence.
The toughness, stability, and resistance to most acids and alkalis of PVDF are well regarded. In comparison to nitrocellulose, PVDF membranes also offer improved protein retention. Nitrocellulose membranes often have high signal-to-noise ratios and are efficiently inhibited. However, you won't be able to strip them, re-probe them, or measure the nitrocellulose-membrane pore size.
Antibody Titration
Their respective concentrations in solution impact how quickly an antibody binds to an antigen. In Western blotting, the amount of the antigen is typically fixed; therefore, you frequently need to adjust the antibody concentration to get better blots. You can find the best antibody concentration for your research by carrying out this procedure methodically, evaluating several antibody dilutions while maintaining all other variables constant.
Blocking
There are numerous blocking buffers, but not all are effective in all circumstances. For each antibody-antigen pair, it is crucial to test various blocking buffers. Milk-based blocking buffers may contain signal-interfering substances, including endogenous biotin, glycoproteins, and enzymes.
Washing
When we've had a strong background signal, going over the washing procedures again on the subsequent run-through has been beneficial. We've mostly accomplished this by raising the Tween-20 concentration from 0.05% to 0.1%.
Moreover, you can change the degree of washing by utilizing strategies such as increased washing volumes and times, more buffer changes, or more potent detergents. Don't go overboard.
Detection
To get a robust Western blot signal, the detection phase is essential. If your existing detection method doesn't fulfill lab requirements, several chemiluminescent reagents are available and chemiluminescent alternatives, such as fluorescence detection. For instance, you can select more sensitive reagents or those that create signals that stay longer. The latter option will significantly improve your blots' repeatability.
Final Words
Optimization of blotting systems aim to lower non-specific bands and boost the signal-to-noise ratio of certain bands (if present). Optimizing all the aforementioned factors will take a lot of time, but even a few little changes could have a significant impact and save you time in the long term.
