#Imagej for western blot quantification plus

They are visible as blue bands during these processes. Dark bands are pre-stained protein used to monitor progress of protein electrophoresis, blot orientation, and assess transfer efficiency. They are detected after incubation with Eu-streptavidin and read on the ScanLater Western Blot Detection System. White bands are biotinylated protein used to estimate molecular weights of blotted proteins.

Eu-streptavidin binds to the protein ladder, and Eu-secondary antibody binds specifically to the primary antibody attached to the protein of interest.įigure 1: ScanLater Western Blot Protein Ladder. After incubation with primary antibody, membranes undergo blocking and washing, followed by incubation with 1:5000 Eustreptavidin and 1:5000 Eu-secondary antibody. 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.

#Imagej for western blot quantification plus

A single set of standards contains markers for both visual gel and blot alignment plus TRF detection.Application Note Estimation of Protein Molecular Weights with ScanLater Western Blot Protein Ladder