#Protein ladders for western blotting plus#

ScanLater Western Blot Protein Ladder was also loaded onto the same gel. Interleukin-17 (IL-17) was loaded on to a 4-20% gradient gel in 1X running buffer and run for 30 minutes. Estimate of molecular weight is approximately 70 kD. Western blot shows an increase in HSP70 that occurs when cells are exposed to temperatures that are higher than normal. (3b) Western blot showing an increase in HSP70.

Estimation of the molecular weight of IL-17 using the ScanLater Western Blot Protein Ladder. Visual estimation of known proteinsįurther experiments with Interleukin-17 and Heat Shock Protein also exemplify the use of the ScanLater Western Blot Protein Ladder for estimating molecular weight.įigure 3: (3a) Molecular weight estimation of IL-17. The linear relationship is similar to that of a reference product from Sigma cat. The migration distances of seven proteins in the ScanLater Western Blot Protein Ladder are plotted and fitted as a function of their respective molecular weights. The ScanLater Western Blot Detection System, including the ScanLater Western Blot Protein Ladder, is a simple, sensitive, and stable platform that provides excellent protein analysis capability in a multi-mode microplate reader.įigure 2: Migration vs. Europium is resistant to photo-bleaching, so the western blot signal remains stable for weeks to months which enables repeated reading of membranes. The method does not involve an enzymatic reaction, and as a result removes the inherent variability for protein quantitation. There is no camera blooming which is often seen with chemiluminescence or standard fluorescence detection thus the system provides sharp bands with high S/N ratio. The TRF detection significantly reduces background noise from auto fluorescence or other sources of short lifetime emissions. Membranes are scanned on either the SpectraMax Paradigm or SpectraMax i3 Multi-Mode Microplate Reader with the ScanLater Western Blot Detection Cartridge,which utilizes time resolved fluorescence (TRF) detection of Europium (Eu). 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.

#Protein ladders for western blotting 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