Protein Characterization

A single band on SDS PAGE is inadequate demonstration of protein purity. The band can be comprised of multiple proteins of similar molecular mass, either post translational variants of the same protein or other totally unrelated proteins. Western blotting alone does not confirm purity.

Focus Proteomics provides detailed characterization of your proteins of interest, from the separation of post-translational modified isoforms by IEF or affinity PAGE to their identification by mass spectrometry.

We use Delta2D advanced image analysis software for statistically significant protein expression analysis.

Figure 1.

Transfer efficiency as a function of protein molecular mass (kDa) and gel concentration as measured from 4-20% (left) and 10% (right) polyacrylamide gels. Protein standards were 315, 250, 180, 140, 95, 55, 42, 26, and 17 kDa (from dark to light). Each protein was run in triplicate on each gel type and transferred under identical conditions. Protein bands were measured in (A) the post-transfer gels and (B) the primary PVDF membrane and expressed as the percent total protein as determined in untransferred control gels. The blow through of proteins was estimated from (C) a secondary membrane situated behind the primary membrane. Over 20% of a 26 kDa protein was blown through the primary membrane.

Focus Proteomics

Provides 1D or 2D PAGE coupled with Western blotting for the in depth characterization of biopharmaceuticals. Multiplex staining compares total protein and immunospecific staining to differentiate the target protein from contaminating HCPs. Carbonylation, a measure of protein oxidation, can also be quantified from Western blots.