2D Western Blot for HCP Coverage Determination
The suitability of HCP antibodies for their specific processes needs to be shown, not only by the ELISA data obtained, but also by determination of the HCP coverage in percent.
The 2D Western blot method is widely accepted for HCP coverage determination. In this method, proteins are separated in two dimensions according to their isoelectric points (isoelectric focusing, IEF) and to their molecular weights (SDS-PAGE) followed by Western blotting and immunostaining.
BioGenes offers fluorescence-based 2D Western blotting, which usually involves fluorescent minimal labeling of the sample proteins and immunodetection using the HCP antibody and a suitable fluorophore-conjugated detector antibody. Therefore, immunostaining can be performed on the same Western blot membrane on which total protein detection also occurs. The fluorescence-based method has replaced the colorimetric determination of HCP coverage due to improved sensitivity and precision. Alternatively, fluorescent total protein staining on a 2D Western blot membrane can also be performed by non-covalent stains to flexibly adapt methodology to the requirements of individual samples.
Fluorescent total protein pattern Sci5-minimal labeling
HCP-specific immunostaining, detection by Cy3-secondary antibody conjugate
Overlay (app. 70% coverage)