New Proteomic Subfractionation Surfaces – Innovative Technology for the Improved Resolution of Serum Proteins
Poster Jan 10, 2006
Swapan Roy, John Krupey,Matthew Kuruc and Devjit Roy
IntroductionProFACT™ is a new subfractionation methodology designed for comparative proteome analysis. Electrophoretic profiles of serum subfractions demonstrate improved resolution and quantification. Carryover from the three highest abundance serum proteins, Albumin, IgG and Transferrin is minimal. The process starts with a separation platform utilizing a new combination of surface microenvironments substituted with low molecular weight substrates that feature drugbinding motifs.
With the ProFACT™ surface library, undenatured, bioactive proteins can be subfractionated into differential pools. Separations are universal as they do not require prequalified binding knowledge, a key limitation of affinity-type techniques. The surfaces utilized are disposable and adaptable to sample size and scale requirements. A simple bind, wash and elute protocol is completed in 30 to 60 minutes and as elutions are mild and consistent, a direct handoff can be made to subsequent interrogation. The interrogation strategy is adaptable to meet investigative inquiry using ‘bottom-up’ or ‘top-down’ approaches.
Detailed iterative profiling can be applied towards biomarker discovery, disease-state pattern identification, systems biology, or otherwise be useful to reduce sample complexity. The ProFACT™ surface library can potentially be coupled with HPLC, Capillary Electrophoresis, 1 and 2D Electrophoresis, and Mass Spectrometry to expand coverage and sensitivity. As structural modifications of proteins can alter their binding affinities to the surface library, structural differences in sample sets may be inferred upon interpretation of ProFACT™ subfraction profiles.
The data presented herein demonstrate the unique profiling capabilities of each ProFACT™ surface library subfraction and the collective resolution of 69 non-redundant proteins, calibrated and quantified from image analysis of SDS-PAGE profiles. 10 differential protein subfractions can be generated in less than 1 hour, without the need for immuno-depletion. Future investigations will focus on comparing normal and disease state sera.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE