Next Generation 2D Electrophoresis

• Next Generation IEF and 2D PAGE
• Characterization of protein biologics and identification of host cell proteins (HCPs)
• Native PAGE, SDS PAGE, and Western blotting
• Phosphoaffinity PAGE
• BAC PAGE and 2D BAC-SDS PAGE of membrane proteins
• Multiplex staining of phosphoproteins, polyhistidine-tagged proteins, and total proteins (SYPRO Ruby, KUMASI, or silver staining)
• Affinity enrichment of extremely low abundance proteins.
• Advanced image analysis for statistically significant protein expression analysis
• Spot excision and trypsin digestion for LC-MS

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Figure 1.

Sample preparation is critical to meaningful 2D results.  Rhodopseudomonas palustris cells were divided equally and one half (left) was processed in 7M urea, 2M thiourea, 25 mM C7BZO and the other half (right) was processed in CytoBuster reagent.   The latter reagent was optimized for the recovery of recombinant proteins, and therefore, exhibits bias towards cytoplasmic proteins.  Membrane proteins are more accurately represented in left gel while cytoplasmic proteins are overrepresented in the right gel.

Figure 2.

Protein expression analysis and statistical significance of image analysis of protein expression.   Volcano plot illustrates the significance of differences in spot fluorescence in replicate 2D gels comparing stabilized (U) and unstabilized (S) phosphoproteins (red).  Fold difference in 595 matched spots in 2D gels were plotted on the abscissa.  Significance is expressed on the ordinate as the negative log of the ANOVA value.  Gel reproducibility (yellow) is represented by comparison of replicate gels and their close adherence to the expected 1:1 ratio.

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