ABSTRACT
We report the establishment of highly non-redundant unigene sets consisting of cDNA clones derived from T lymphocytes and natural killer cells. Each set consists of 10 506 and 13 409 clones, respectively, arrayed on nylon membranes in duplicate. The sets provide an excellent tool for genome-wide gene expression analysis studies in immunology research.
Subject(s)
Gene Expression/physiology , Killer Cells, Natural/metabolism , Leukemia/genetics , Leukemia/metabolism , T-Lymphocytes/metabolism , Blotting, Northern , DNA, Complementary/metabolism , Gene Expression Profiling , Gene Library , Humans , Jurkat Cells , MaleABSTRACT
The large-gel two-dimensional electrophoresis (2-DE) technique, developed by Klose and co-workers over the past 25 years, provides the resolving power necessary to separate crude proteome extracts of higher eukaryotes. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) provides the sample throughput necessary to identify thousands of different protein species in an adequate time period. Spot excision, in situ proteolysis, and extraction of the cleavage products from the gel matrix, peptide purification and concentration as well as the mass spectrometric sample preparation are the crucial steps that interface the two analytical techniques. Today, these routines and not the mass spectrometric instrumentation determine how many protein digests can be analyzed per day per instrument. The present paper focuses on this analytical interface and reports on an integrated protocol and technology developed in our laboratory. Automated identification of proteins in sequence databases by mass spectrometric peptide mapping requires a powerful search engine that makes full use of the information contained in the experimental data, and scores the search results accordingly. This challenge is heading a second part of the paper.
Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Data Collection , Data Display , Databases, Protein , Electrophoresis, Gel, Two-Dimensional/instrumentation , Eukaryotic Cells/chemistry , Peptide Mapping , Plant Proteins/analysis , Plant Proteins/isolation & purification , Proteins/isolation & purification , Proteome , Specimen Handling/instrumentation , Specimen Handling/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentationABSTRACT
The usage of standard 96 well microplates for the screening of crystallization conditions of recombinant proteins offers several advantages when compared to commonly used crystallization plate formats. The adoption of robotic technology for plate and glass slide preparation within a "hanging drop" vapour diffusion crystallization experiment enables to work with an increased throughput at reduced costs. In addition to commercial pipetting devices with a 96-channel aspirator/dispenser, solenoid ink-jet technology was applied to form 250 nl droplets with a diameter of approximately 1 mm. This allows miniaturization of crystallization screening set-ups with an estimated ten-fold cost reduction when compared to commonly used 24 well plates.