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Sample Preparation Solutions for Cellular Protein Fractionation Poster, Rev A

ware and spots of interest were cut from the gels using the ProteomeWorks spot cutter. Peptide mass fingerprint data was obtained from excised 2-D gel spots using the MassPREP station robotics system for protein digestion and MALDI target spotting. Gel spots were destained, reduced, alkylated, dried, digested with modified trypsin (Promega) while heated at 37C for 5 hr, and extracted with an acidic solution. Peptides were then automatically spotted onto MALDI target plates mixed with a matrix solution of 2 mg/ml -cyano-4-hydroxycinnamic acid. MALDI mass spectra were acquired on a M@LDI-LR instrument in reflector mode. A nitrogen laser (= 337 nm, Laser Science) was pulsed at 20 Hz. Ions were accelerated to 15 kV after a timelag focusing pulse of 2,750 V at 500 ns. Data were collected from the sample well and a near point lock mass (ACTH clip 1839, Sigma). The MALDI mass spectra were processed to identify monoisotopic peaks using the Micromass algorithm MaxEntLite. The search engine used to identify peptide mass fingerprint data was ProteinLynx Global SERVER 2.0 software.

Results
Three separate sample preparation protocols were used to fractionate and enrich mouse liver protein samples prior to 2DE. Although the proteins for each gel were prepared from the same starting material, the final gel results following fractionation provide completely different views of the mouse liver proteome (Figure 1). Proteins that were unique to the fractionated sample were selected for identification. Table 1 shows selected proteins from these gels that are associated with the cellular structures enriched for in the sample separation protocols. Many of these identified proteins are membrane associated.
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Applications: Epitope mapping, Mapping protein-protein contacts, Identification of enzyme substrates/inhibitors, Identification of receptor agonists/antagonists...
Bethyl Laboratories packages antibodies, conjugates and calibrators to provide quantitative ELISA kits. Each kit contains the following components, sufficient for 1000 single well assays....
Bethyl Laboratories packages antibodies, conjugates and calibrators to provide quantitative ELISA kits. Each kit contains the following components, sufficient for 1000 single well assays....
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