Two-dimensional gel electrophoresis is being used to purify and characterize the expressed proteins in E. coli for comparing their observed N-terminus, pI, MW, abundance, and subcellular location to predictions from the DNA sequence. Total cell extracts have been prepared by either SDS-heat or sonication protocols. For detection by Coomassie-Blue and protein sequencing, 200-250 mg of protein in a volume less than 40 ml is typically loaded onto the first dimension IEF gel. Keeping the final SDS concentration of the protein extract below 0.3% was important to prevent loss of resolution during IEF. To identify protein cellular location and to enrich for low abundance proteins, cellular fractionation based on subcellular location is being used. Using an osmotic shock method, proteins located in the periplasmic space have been enriched approximately 30-fold compared to the total extract. After the second dimension SDS-PAGE, proteins are electrotransfered onto PVDF in 10 mM CAPS, pH 11.0, 10% methanol buffer and detected by staining with Coomassie-Blue. No proteins were detected in the second dimension gel which did not transfer. Most E. coli proteins are unblocked and can be sequenced directly from PVDF by Edman degradation. From 388 spots, 299 unique amino-termini and isoforms have been found. The N-terminal sequence and observed pI, MW, and abundance of the protein has been used to re-interpret the predicted open reading frames of the genomic sequence.
From Analytical 2-D Gels to Identification of Spots from Preparative 2-D Gels, Scott D. Patterson.
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has long been recognized as a powerful tool for the analysis of biological samples. When combined with a means to quantitatively analyze these patterns, 2-D PAGE provides the ability to analyze complex patterns of gene regulation both temporally and spatially (i.e., subcellular enrichment). However, the first step to achieving this aim is being able to run reproducible 2-D gels. This requires careful attention to sample preparation, first- and second-gel dimension preparation and running, as well as reproducible protein detection. The approaches used to obtain reproducible 2-D PAGE patterns or radiolabeled Jurkat T-lymphoblasts for subsequent quantitative analysis will be addressed. Once protein spots of interest have been revealed from the quantitative analysis of the 2-D PAGE patterns, the problem of identification is then encountered. Two of a number of approaches for this will be covered, immunoblotting and peptide-mass fingerprinting. With the advent of many 2-D PAGE databases there are a number of protein spots that are already "identified" in a few cell lines. Combined with the aims of the experiment, these databases may give one the opportunity to guess at the identity of a particular protein spot and confirm or deny this by immunoblotting. The approach of obtaining accurate peptide masses from specifically cleaved proteins to search protein sequence databases, known as peptide mass fingerprinting, provides one with another opportunity to identify a previously sequenced protein or (hopefully) confirm that it is indeed novel. These methods will be addressed, including such issues as sample preparation for mass spectrometry following various enzymatic digestions.
Amino Acid Composition-A Robust and Flexible Identification Tool for Proteins Separated by 2-D Electrophoresis, M. Wilkins, I. Humphery-Smith, D. Hochstrasser, Y. Fung, M. Duncan, N. Packer, K. Williams, and A. Gooley.
We have been using amino acid analysis for 18 months to identify proteins from 2-D gels. We will give an overview of what can be achieved in the areas of single species matching and cross-species matching and illustrate how amino acid analysis can be used in conjunction with other techniques to achieve protein identification and characterisation. Examples will be shown from E. coli, Mycoplasma genitalium, and human plasma.
Construction and Analysis of Two-Dimensional Gel Protein Databases, Jerry Latter.
There are several software systems available for the construction or analysis of two-dimensional (2D) gel databases. This talk will cover the existing software systems and how they can be used to analyze an experiment or to create a 2D gel database. The current status of 2D gel databases will be described as well as the ability to access 2D gel information over the Internet. Topics in 2D gel analysis will include data acquisition, data linearity, image processing before analysis, and analysis techniques. Potential pitfalls will be discussed as well as when computer analysis is beneficial.
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