#Western blot protein size ladder verification

Rustandi RR, Anderson CL, Hamm M (2013) Application of capillary electrophoresis in glycoprotein analysis. Rustandi RR, Wang Y (2011) Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress. Rustandi RR, Washabaugh MW, Wang Y (2008) Application of CE SDS gel in development of biopharmaceutical antibody-based products. Ma S, Nashabeh W (2001) Analysis of protein therapeutics by capillary electrophoresis. Hunt G, Nashabeh W (1999) Capillary electrophoresis sodium dodecyl sulfate nongel sieving analysis of a therapeutic recombinant monoclonal antibody: a biotechnology perspective. Koller A, Wätzig H (2005) Precision and variance components in quantitative gel electrophoresis. Ghosh R, Gilda JE, Gomez AV (2014) The necessity of and strategies for improving confidence in the accuracy of Western Blots. Kurien BT, Scofield RH (2006) Western blotting. Renart J, Reiser J, Stark GR (1979) Transfer of proteins from gels to diazobenzyloxymethyl paper and detection with anti-sera: a method for studying antibody specificity and antigen structure. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to NC sheets: procedure and applications. Details of sample preparation and experimental conditions for each capillary western mode are described in this chapter. difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western techniques. Examples using this technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C. The size-based capillary western can be used for fast antibody screening, clone selection, product titer, identity, and degradation while the charge-based capillary western can be used to study product charge heterogeneity. We have used this technology for both charge- and size-based CE westerns to analyze biotherapeutic and vaccine products. Here, we describe the applications of a relatively new CE-based western blot technology which is automated, fast, and quantitative. This technique is widely used across industrial and academic laboratories, but it is very laborious, manual, time-consuming, and only semi-quantitative.

Another important and traditional slab gel technique is the western blot, which detects proteins using immuno-specific reagents after SDS-PAGE separation. These two CE-based methods are now an industry standard and are an expectation of the regulatory agencies for biologics characterization. In recent years, labor-intensive SDS-PAGE and IEF slab gels have been replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical industry. Under suggested conditions, BLUeye Prestained Protein Ladder resolves 12 major bands in 15 % SDS-PAGE (Trisglycine buffer) and after Western blotting to nitrocellulose membrane.Īpproximately 0.1~0.4 mg/mL of each protein in the buffer (20 mM Tris-phosphate, pH 7.5 at 25 ☌), 2 % SDS, 1 mM Dithiothreitol, 3.6 M Urea, and 15 % (v/v) Glycerol).Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied to characterize proteins. The ladder is supplied in gel loading buffer and is ready to use.

#Western blot protein size ladder verification

The BLUeye Prestained Protein Ladder 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 a blue chromophore except for two reference bands (one green and one red at 25 kDa and 75 kDa respectively) when separated on SDS-PAGE (Tris-glycine buffer). The BLUeye Prestained Protein Ladder is a three-color protein standard with 12 pre-stained proteins covering a wide range molecular weights from 10 to 245 kDa. 1.5-2.5 μL per well for general Western transfering.īLUeye Prestained Protein Ladder resolves 12 major bands in 15 % SDS-PAGE (Tris-glycine buffer) and after Western blotting to nitrocellulose membrane. Use 3 μL or 5 μL per loading for clear visualization during electrophoresis on 15-well or 10-well mini-gel, respectively.

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