With the popularity of fluorescence detection, many researchers have turned western blot detection from chemiluminescence to multiple fluorescence. This is because fluorescence detection enables multiple western blot analysis, enabling simultaneous detection of several target proteins at a time without the need for stripping and rehybridization. In addition, the benefits of fluorescence are a wider dynamic range and better signal stability.
In order to make your experiment process easy and successful, the experts of Deep Blue Cloud Biological Research Department brought us some tips for fluorescent western blot.
★ Basic knowledge of fluorescent Western Blot ★
Fluorescent western blots can perform multiple assays and generate quantitative and accurate data to meet the journal's requirements for data publication. Fluorescence detection solves some of the limitations of chemiluminescence detection, such as data reproducibility, semi-quantitative, dynamic range and other issues. Therefore, fluorescent Western blots are becoming more and more popular. Fluorescence methods are a good choice for researchers who need more sophisticated linear analysis of signals and protein abundance correlations.
Once you decide to use a fluorescent digital imaging system, the next step is to carefully consider the imprinting reagent. High quality, low fluorescence membranes are the first choice for fluorescent Western blotting. It is also necessary to optimize the blocking solution and rinse solution to provide a better solution. One of the biggest problems encountered by small partners in switching from chemiluminescence to fluorescence detection is the high background. Using the right membrane, as well as the blocking solution and rinse solution, will ensure that you start the correct fluorescent western experiment. In the transfer process, the key is to use a protocol that does not generate too much heat. High temperatures can cause backgrounds to rise, especially in visible fluorescence detection. Therefore, in a buffer that does not generate excessive heat, it is an ideal choice to perform wet or semi-dry rotation in a short time.
The final and most challenging reagent selection is the choice of fluorescent secondary antibodies. Fortunately, with the increasing popularity of fluorescent detection methods, there are a large number of commercially available fluorescent secondary antibodies available on the market. Before selecting an antibody dye conjugate, it is important to understand the excitation and emission wavelengths of each channel of the imaging system. Once you understand this information, you can choose the dye that is most compatible with your system.
How to improve the effect of fluorescent western blot detection
★ Determine the original concentration of primary and secondary antibodies by titration.
★ Optimize the target protein and internal reference separately before multiple detection.
★ The concentration of primary antibody used is higher than that of traditional chemiluminescence 2-5X. The concentration of the secondary antibody also needs to be increased. It is generally recommended to use an initial dilution of 1:5000.
★ Use a low background PVDF membrane. The NC film and some PVDF autofluorescence are high, causing a high background.
★ Avoid the fluorescence generated by the mark and the dye itself. Mark the blotting membrane with a pencil. For example, bromophenol blue and Coomassie brilliant blue have autofluorescence.
★ Keep it clean, wash the trays and other equipment before use, wear gloves to treat the gel, and treat the membrane with tweezers.
★ Fluorescent antibodies should be stored away from light to prevent quenching and affect the use of antibodies.
★ When performing multiple tests, the dye should be avoided to overlap and the dye with a longer spectral distance should be selected for incubation.
★ If you want to save the blotting film, you need to keep it in the dark.
How about, after reading the western blot tips above, are you already confident and full of fighting spirit? Don't worry, if you want to do something good, you must first sharpen your tools. The stable and perfect Western Blot, in addition to relying on the actual station experience, needs the support of classic experimental products. Classic products are classic because they have been proven by countless researchers. Deep Blue Cloud not only integrates the classic products of the key steps of Western Blot, but also provides the best solution for your experiments, supporting your Western Blot step by step. Every time, it can be done elegantly and quickly! Small partners who need it can contact us as follows.
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