#Protein ladders for western blotting verification#
#Protein ladders for western blotting plus#

#Protein ladders for western blotting 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. It is concluded that the estimated molecular weight of SMOBIO’s pre-stained marker shows a curve matching well with that of unstained native proteins (MARK12), representing a good estimation of the MW of each pre-stained protein in the SDS-PAGE analysis.Choosing the right gel is a key factor in the successful transfer of HMW proteins. Although it is impossible to define 'precision' for molecular weight of proteins in SDS-PAGE, we did compare the migration pattern of pre-stained markers with unstained protein marker (Invitrogen MARK12) for calibration. Therefore, in the product description, we suggest our users to calibrate the MW against their interested proteins. We did compare the migration patterns of SMOBIO’s Protein Markers with other brands, and we concluded that it was difficult to define “precision” due to the reasons mentioned above. For example, a protein which is highly hydrophilic might show a particular higher position in the SDS-PAGE analysis when compared to a hydrophobic one. It is known that the analysis of protein size by an SDS-PAGE is only for “estimation” because of the intrinsic variation of amino acid composition in all proteins including stained and non-stained ones. Usually, pre-stained marker is written on “estimated molecular weight” for caution. MOPS, and MES buffer) and after Western blotting to nitrocellulose membrane. Under suggested conditions, PM5200 ExcelBand™ 3-color Pre-Stained Protein Ladder Broad Range resolves 15 major bands in SDS-PAGE ( Tris-Glycine, Three reference bands - 75 kDa (red), 40 kDa (green), and 20 kDa (blue)Īpproximately 0.1~0.4 mg/ml of each protein in the buffer (20 mM Tris-phosphate (pH 7.5 at 25☌), 2% SDS, 0.2 mM DTT, 3.6 M urea, and 15% (v/v) glycerol). Ready-to-use - Premixed with a loading buffer for direct loading, no need to boil.

#Protein ladders for western blotting verification#

The PM5200 3-color Pre-Stained Protein Ladder Broad Range is designed for monitoring protein separation during SDS-polyacrylamide gel electrophoresis, verification of Western transfer efficiency on membranes (PVDF, nylon, or nitrocellulose) and for approximating the size of proteins. Proteins are covalently coupled with different chromophores for easy identification of bands, with three reference proteins carrying enhanced intensity corresponding to a blue band at 20 kDa, green at 40 kDa, and red at 75 kDa, respectively, as separated on SDS-PAGE (Tris-Glycine buffer).

The PM5200 3-color Pre-Stained Protein Ladder Broad Range is a ready-to-use three-color protein standard with 15 pre-stained proteins covering a wide range of molecular weights from 5 to 245 kDa in Tris-Glycine Buffer (3.5 to 235 kDa in Bis-Tris (MOPS) buffer and Bis-Tris (MES) buffer).