Jacek Mozdzanowski

Electroblotting from Polyacrylamide Gels

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N‐terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications.

Two‐Dimensional Gel Electrophoresis

Two‐dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two‐dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS‐PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two‐dimensional gels, and two‐dimensional protein databases.

High Sensitivity Peptide Sequence Analysis Using In Situ Proteolysis on High Retention PVDF Membranes and a Biphasic Reaction Column Sequencer

Publisher Summary This chapter explores the high-sensitivity peptide sequence analysis using in situ proteolysis on high-retention polyvinylidene difluoride (PVDF) membranes and a biphasic reaction column sequencer. In a study described in the chapter, comparative high-performance liquid chromatography (HPLC) peptide maps of transferrin and myoglobin were used to evaluate relative yields of tryptic peptides from different types of blotting membranes. The recovery of most tryptic myoglobin peptides from Trans-Blot PVDF is three to five times higher than from nitrocellulose. The observed differences in yields between membrane types appear to primarily reflect differences in electroblotting recoveries rather than variations in proteolysis or peptide extraction because the observed peptide yields on the HPLC profile roughly correlate with the blotting efficiency on the different membrane types. In addition, only minor differences in peptide recoveries from different membrane types were observed for transferrin, which is well retained by all membrane types evaluated. High-retention PVDF membranes such as Trans-Blot are the preferred membranes for routine in situ protease digestions of gel purified proteins because of their high retention of proteins during electroblotting.

Gamma-Interferon-Induced Nerve Growth Factor Receptors in Colorectal Carcinoma Cell Lines

Gamma-interferon (gamma IFN) was found to induce expression of the 150,000 M(r) cell surface and the 35,000 M(r) chromatin receptors for nerve growth factor (NGF) in the SW1116 colorectal carcinoma cell line that does not express NGF receptors. In the SW707 colorectal carcinoma cell line that expresses a low level of NGF receptors, gamma IFN stimulated expression of the cell surface and the nuclear receptors. Induction of NGF receptors in SW1116 cells resulted in internalization and nuclear translocation of 125I-NGF. When NGF bound to the chromatin, ribosomal RNA synthesis was inhibited. Two-dimensional gel electrophoresis of [35S]methionine-labeled chromatin proteins indicated significant changes in chromatin protein composition in cells treated and not-treated with gamma IFN. gamma IFN effectively stimulated the expression of NGF receptors in two colorectal carcinoma cell lines, but inhibited the expression in melanoma and breast carcinoma cells. It is suggested that gamma IFN, by modulating the expression of NGF receptors may affect the NGF-dependent growth of some tumor cell lines.

UNIT 10.7 Electroblotting from Polyacrylamide Gels

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications.

Protein Sequence Analysis Using Hewlett-Packard Biphasic Sequencing Cartridges in an Applied Biosystems 473A Protein Sequencer

Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.g., formulated protein drugs) can be easily analyzed using the ABI sequencer. Simple modifications to the ABI sequencer to accommodate the cartridge extend its utility in the analysis of difficult samples. The ABI sequencer solvents and reagents were compatible with the HP cartridge for sequencing. Sequence information up to ten residues can be easily generated by this nonoptimized procedure, and it is sufficient for identifying proteins by database search and for preparing a DNA probe for cloning novel proteins.