As a result, fluorescent blotting provides quantitative and consistent results across repeat experiments. The fluorophore emits light only when excited with light of the appropriate wavelength. 2 Table 1 below outlines the main differences between chemiluminescent and fluorescent western blotting.Īs mentioned in Table 1 above, fluorescent western blotting uses a secondary antibody labeled with a fluorophore. However, the signal obtained in fluorescent blotting is a static value proportional to the abundance of target proteins in complex with fluorophore-bound antibodies. In chemiluminescent blotting the signal obtained is variable and generally regarded as semi-quantitative. Fluorescent Western Blotting – Here Comes the Science Part! That is, a move from chemiluminescent to fluorescent detection. The improved precision is partly down to improved detection methods. This is because even if the primary or secondary blotting antibodies cross react with non-target proteins, the signal you want (i.e., target antigen) can usually be differentiated from the noise (non-specific binding) based on molecular weight.Īdvances in reagent and imaging technologies have moved western blotting from a qualitative assessment of target proteins to a quantitative measurement of target protein levels, which is useful in broad fields including cellular biology and antibody research. Protein separation by electrophoresis followed by western blotting may offer more precision than other antibody-based detection assays (e.g., ELISA). Importantly, this technique can save you time and precious samples.įirst, let’s have a quick recap! Western blotting is an efficient technique to assess the presence and abundance of proteins of interest. Lastly, we’ll look at the many advantages of fluorescent western blotting, including the possibility to multiplex. Then, we will learn how infrared fluorescent western blotting can give you truly quantitative and reproducible results. Firstly, we will compare fluorescent and chemiluminescent western blotting. So, you choose! Then, experience the sharp, vibrant colors of LI-COR Chameleon Pre-stained Protein Ladders in your near-infrared fluorescent Western blot applications.In this article, you will be introduced to the world of fluorescent western blotting. Chameleon 800 Pre-stained Protein Ladder for analysis in the 800nm channel.Chameleon 700 Pre-stained Protein Ladder – for 700nm channel analysis. Of course, if you are doing just one-channel analysis of your Western blot, you can try the.Or, if you would like to mix your own to customize fluorescence intensities and are doing multiplex detection, try the Chameleon Kit Pre-stained Protein Ladder (includes 250 μL each of the Chameleon 700 and the Chameleon 800 Pre-stained Protein Ladders).This Chameleon Ladder is a pre-mixed protein MW marker format– ready to use! Try the Chameleon Duo Pre-stained Protein Ladder if you are doing two-color infrared fluorescent detection.With them, you can easily identify gel migration and protein size and orient your gel and membrane quickly. These pre-stained ladders are multi-colored for easy molecular weight identification. And, if you need pre-stained protein molecular weight ladders that you can see AND detect with near-infrared fluorescence, then you need Chameleon ® Pre-stained Protein Ladders. If you are doing Western blots, then you are most likely using a protein ladder.
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