Licor blocking buffer12/16/2023 ![]() ![]() ![]() Experimental details are identical to those provided in Figure 1 above. The settings for all images are linked for accurate visual comparison in Image Studio™ Software. Odyssey Fc (2 minutes) images shown in the Chemi channel. Membrane performance can vary between manufacturers. Protein Electrotransfer Methods and the Odyssey Infrared Imaging System ( LI‑COR Biosciences) provides additional information for optimizing protein transfer. Protein characteristics, such as molecular weight, amino acid composition, and post-translational modifications (e.g., glycosylation or phosphorylation) can alter the transfer efficiency and, ultimately, detection sensitivity. ![]() Detection sensitivities can be greatly affected by your choice of membrane, and you should evaluate multiple membranes. Panel B in Figure 1 shows only the Chemi channel images and highlights protein binding capacity and detection sensitivity of these two membranes.įigure 2 compares Western blot performance on a wide selection of PVDF membranes. These image overlays capture the molecular weight marker signal in the 700 nm channel and the target signal from the Western blot in the Chemi channel. All images shown with normalized image display settings. Image overlays of the 700 and Chemi channel are shown in Panel A Chemi channel only is shown in Panel B. The blots were incubated with SuperSignal® West Dura (Thermo Scientific) and immediately imaged on the Odyssey® Fc Imager for two minutes in both Chemi and 700 nm channels. The membranes were blocked with skim milk, probed with rabbit ß-actin antibody (LI-COR, P/N 926-42211), and detected with Goat Anti-Rabbit-HRP. Two-fold dilutions of C32 (156 μg - 1.25 μg) lysate were resolved on a 10% Bis Tris gel and transferred to the indicated PVDF membranes. A comparison of protein binding capacity and detection sensitivity of two membranes. Panel A in Figure 1 highlights the background differences between two PVDF membranes in the 700 nm channel. Sensitivity is affected by many factors, including protein transfer efficiency, protein binding capacity, and membrane autofluorescence–particularly when visualizing blue molecular weight marker bands in the 700 nm channel. Your choice of membrane is critical to the success of a chemiluminescent Western blot. Incubate with HRP- or AP-conjugated secondary antibodyĭetect with an Odyssey M Imager, Odyssey XF, Odyssey Fc, or C‑DiGit Blot Scannerįactors that Affect a Chemiluminescent Western Blotīelow are some things that can affect the results you receive from a chemiluminescent Western blot. Wet the membrane according to membrane type (nitrocellulose or PVDF) Transfer to a membrane (nitrocellulose or PVDF) Chemiluminescent Western Blot Workflowĭetermine protein concentrations and prepare samples This additional capability enables the use of fluorescent markers on the same blot as the chemiluminescent targets. The C‑DiGit Blot Scanner is a chemiluminescent detection system only, whereas the Odyssey Fc, Odyssey XF, and Odyssey M are capable of fluorescent detection, as well. The following LI-COR imagers support chemiluminescence imaging and other detection labels.Ĩ00, 700, 520, 488, Epi (RGB, 630, 525, 470), Trans (RGB, 630, 525, 470) Photographic film or an imaging system using a digital CCD camera captures the emitted light as an image. A chemiluminescent substrate for the enzyme is carefully applied to the blot, and light is emitted when the enzyme modifies the substrate. Then the membrane is probed with a detection antibody or conjugate.Ĭhemiluminescent Western blots are probed with a primary antibody against the target protein, followed by a secondary antibody labeled with an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The membrane is blocked to prevent non-specific antibody binding. Next, the proteins are transferred to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. ![]() First, proteins are separated using gel electrophoresis. It is now a common laboratory technique with many variations. in 1979 as a simple method to electrophoretically blot proteins onto nitrocellulose sheets. Western blotting was introduced by Towbin, et al. Maximizing sensitivity on chemiluminescent Western blots Introduction Optimizing Chemiluminescent Western Blots ![]()
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