No heating or other preparation of the ladder is required. Along with protein samples of interest, simply load one of the lanes on the gel with 4 µL of ScanLater Western Blot Protein Ladder. The ScanLater Western Blot workflow follows standard gel loading and blotting methods up to the secondary antibody incubation step. The linear relationship between the molecular weight of the biotinylated ladder proteins and their migration distance on the gel is examined in Figure 2 in comparison with a reference standard. They are also seen as dark bands when the ladder is read with the Scanlater Western Blot Detection System. These blue bands are seen on the electrophoresis gel and on the blot after transfer. In addition, there are three visible pre-stained marker proteins at 18, 31, and 70 kD (Figure 1, right). They are detected by incubating the blot with Europium-labeled streptavidin and they show as light colored bands when read with the ScanLater Western Blot Detection Cartridge (Figure 1, left). The ScanLater Western Blot Protein Ladder consists of seven biotinylated recombinant proteins at 10, 20, 40, 50, 80, 100 and 140 kD. The blot is read with the ScanLater ™ Western Blot Detection Cartridge on the SpectraMax ® i3 or SpectraMax ® Paradigm ® Multi-Mode Microplate Readers. Detection of the biotinylated proteins is enabled by adding Europium-labeled streptavidin to the blot at the secondary antibody step. The S canLater Western Blot Protein Ladder consists of seven biotinylated recombinant proteins as well as three prestained marker proteins. The primary applications of this protein ladder include the estimation of protein molecular weight, the visualization of gel electrophoresis, and the evaluation of the transfer process from gel to blot. The ScanLater ™ Western Blot Protein Ladder is an essential component of the ScanLater ™ Western Blot Detection System. Molecular weights of detected proteins can be estimated.A single set of standards contains markers for both visual gel and blot alignment plus TRF detection.For most researchers, we generally recommend:Ī) ab116027 for proteins between 10 and 180 kDaī) ab116028 for larger proteins up to 245 kDaĬ) ab116029 for smaller proteins down to 3.5 kDaĭ) ab116029 or ab234592 for studying smaller and larger proteins at the same time.Application Note Estimation of Protein Molecular Weights with ScanLater Western Blot Protein Ladder When choosing the right pre-stained protein ladder, the most important choice is based on the size of your protein of interest. Prestained ladders also can use highlight bands of a different color, to make it easier to identify different size bands. Protein ladders are most convenient to use when they are supplied ready to use, with no heating, dilution, or addition of reducing agent required before loading onto the gel. Unstained protein ladders are more accurate for sizing proteins, as the dyes used in prestained ladders can slightly distort the apparent size of the protein ladder proteins on the gel. However, an unstained protein ladder can only be visualized following staining with Coomassie or a similar non-specific protein stain. Using an unstained protein ladder is very useful when you need to accurately calculate the size of your protein. calculate the approximate size of your protein by overlaying an image of the membrane with the ladder with the image generated by antibody staining. verify transfer efficiency between gel and membrane (PVDF, nylon, or nitrocellulose) monitor protein separation during SDS-polyacrylamide gel electrophoresis Using a prestained protein ladder when running a western blot helps when you need to:
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