Poster: Proteomics

Abs # 971: ZOOM® Benchtop Proteomics™ 2DE analysis of the spinach chloroplast proteome utilizing narrow pH range IPG strips

Presenter:	Beardslee, Tom , thomas.beardslee@invitrogen.com
Authors	Beardslee, Tom  (A)   Lindsey, Adam  (A)   Diller, Tom  (A)   Scott, Brad  (A)   Helms, Cinnamon  (A)   Rooney, Regina  (A)
Affiliations:	(A): Invitrogen Life Technologies

High quality two-dimensional electrophoresis (2DE) of plant samples has historically been problematic. Techniques for sample preparation and solublization have been revised to accommodate the complex problems associated with 2DE of plant tissue samples. The objectives of this work were to continue improvements in 2DE of plant samples by simplification of the isoelectric focusing (IEF) step and to illustrate the power of sample-complexity reduction coupled with IEF using narrow pH range IPG strips. Isoelectric focusing was performed in the ZOOM® IPGRunner™ that utilizes disposable cassettes for both rehydration and focusing of IPG strips without oil. The electrical efficiency of the ZOOM® IPGRunner™ allows for lower maximum voltages to be used and complete focusing in as little as ~1800Volt-hours. Spinach leaf extract and spinach chloroplast extract were used in 2DE with 7cm IPG strips of pH ranges 3-10, 4-7, 4.5-5.5, 5.3-6.3, and 6.1-7.1. The three narrow pH range IPG strips used were selected to zoom in on the regions with the highest abundance of proteins in the proteome. This method of zooming in with IPG strips of increasingly narrow pH ranges allows for increased resolution of protein spots with very similar isoelectric points and molecular weights. In addition, sample complexity reduction via organelle isolation improves resolution by enriching the sample for desired proteins and removal of interfering protein spots.