seeblue protein ladder Ctbp1 western blot

#SEEBLUE PROTEIN LADDER CTBP1 WESTERN BLOT PLUS#

Figure 3 demonstrates the increased transfer efficiency of keyhole limpet hemocyanin (KLH), a ~360–400 kDa protein, when the gel was equilibrated with 20% ethanol prior to transfer. To improve transfer efficiency, submerge the gel in 20% ethanol (prepared in deionized water), and equilibrate 5–10 minutes at room temperature on a shaker prior to transfer. Heat generated during the electrophoresis step can cause certain gels to expand alcohol can help shrink the gel to its final size. In addition, the alcohol equilibration step allows the gel to adjust to its final size before transfer. Equilibrating the gel in alcohol removes contaminating electrophoresis buffer salts and prevents an increase in the conductivity of the transfer, which can increase the amount of heat generated. If using a gel chemistry other than Tris-acetate, adding a quick alcohol equilibration step before transfer can greatly enhance the transfer of HMW proteins when not using the ideal gel chemistry. A32735) at a dilution of 1:5,000 for one hour at room temperature.

#SEEBLUE PROTEIN LADDER CTBP1 WESTERN BLOT PLUS#

After overnight incubation, the membranes were washed in TBST and probed with Goat anti-Rabbit (H+L) Highly Cross-Adsorbed Secondary Antibody, conjugated to Alexa Fluor Plus 800 (Cat. 37565) at room temperature and then probed overnight at 4☌ with an EGFR polyclonal antibody (Cat. Membranes were blocked for 30 minutes with Blocker FL Fluorescent Blocking Buffer (Cat. Proteins were transferred with the iBlot 2 Gel Transfer Device onto nitrocellulose membranes (Cat. EA03752BOX) or onto a Novex 4–20% Tris-glycine gel, WedgeWell format (Cat. Method: Western blot analysis of EGFR was performed by loading serially diluted A431 cell lysate onto a NuPAGE 3–8% Tris-acetate gel (Cat. Western blotting analysis of EGFR expression in A431 lysates transferred from an Novex 4–20% Tris-glycine gel and a NuPAGE 3–8% Tris-acetate gel using the iBlot 2 Gel Transfer Device. As seen in Figure 2, better transfer is seen using a Tris-acetate gel over a 4–20% Tris-glycine gel-9 ng visualized when a Tris-acetate gel was used vs 750 ng visualized when a Tris-glycine gradient gel was used in targeting ~190 kDA protein epidermal growth factor (EGFR).įigure 2. A comparison of HMW protein separation using different gel chemistries and gradients shows the best separation and resolution of HMW proteins can be accomplished with a 3–8% Tris-acetate gel ( Figure 1B). The open matrix structure that allows the HMW proteins to migrate farther through the gel allows better transfer of the HMW proteins out of the gel leading to increased transfer efficiencies and higher sensitivity.

By using a Tris-acetate or low gradient Bis-Tris or Tris-glycine gel, HMW proteins can migrate further through the gel, allowing increased distance between protein bands.

Proteins >200 kDa are compacted into a very narrow region at the top of the running portion of the gel, leading to poor resolution of protein bands ( Figure 1). While 4–20% Tris-glycine gradient gels are very popular because of their ability to separate a broad range of proteins (20–200 kDa), they are not recommended for separation of HMW proteins. When targeting HMW proteins for your transfer, it is best to use a Tris-acetate gel or low percentage Bis-Tris or Tris-glycine gel. Compatible with chemiluminescent substrates and fluorescent secondary antibodies.Choosing the right gel is a key factor in the successful transfer of HMW proteins. Western blotting: detection of the two unstained bands via the detection method used for the target protein.Western blot confirmation-detect the two unstained proteins (30 kDa and 80 kDa) using the detection system for the target protein.Fluorescent visualization-detect the 10 stained proteins using the 670 nm red laser or 700 nm channel of a fluorescence imager.The protein ladder is supplied in a ready-to-use format for direct loading onto gels no need to heat, reduce, or add sample buffer prior to use.Ĭompare and view all other protein standards and ladders › IBright Prestained Protein Ladder contains twelve recombinant proteins, ten (11 to 250 kDa) that are blue-stained and fluor-labeled for direct and near-IR fluorescent visualization and protein sizing, and two (30 kDa and 80 kDa) that are unstained and contain IgG binding sites to bind the primary and secondary antibodies used for chemiluminescent and fluorescent detection of the target protein.

Seeblue protein ladder Ctbp1 western blot