A simple procedure for the isolation of protein disulphide-isomerase.

A two-step procedure is described for the purification of protein disulphide-isomerase (PDI). This procedure is based on the previous finding that the beta-subunit of the prolyl 4-hydroxylase tetramer (alpha 2 beta 2) is identical with PDI [Koivu, Myllylä, Helaakoski, Pihlajaniemi, Tasanen & Kivirikko (1987) J. Biol. Chem. 262, 6447-6449; Pihlajaniemi, Helaakoski, Tasanen, Myllylä, Huhtala, Koivu & Kivirikko (1987) EMBO J. 6, 643-649]. The procedure involves purification of the prolyl 4-hydroxylase tetramer by a simple affinity chromatography and subsequent isolation of the beta-subunit from the dissociated tetramer by ion-exchange chromatography.     

Use of protein biotinylation in vivo for chromatin immunoprecipitation

We describe a system designed to express biotinylated proteins in mammalian cells in vivo and its application to the study of protein-DNA interactions in vivo by chromatin immunoprecipitation (ChIP). The system is based on coexpression of the target protein fused to a short biotin acceptor domain together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction allows one to employ more stringent washing conditions in the ChIP protocol, resulting in a better signal/noise ratio.     

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody–antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.     

Cycled DNA immunoprecipitation procedure to enrich the target sequences for DNA binding proteins with the fold purification monitored.

Using centromere DNA binding protein (CENP-B) expressed as a fusion to beta-galactosidase in Escherichia coli, we established a cycled DNA immunoprecipitation procedure for enriching CENP-B binding sequences and monitoring the enrichment process. Degenerated synthetic oligonucleotides for an authentic CENP-B binding sequence, inserted into a pUC-derived vector, were incubated with the crude CENP-B extract. DNA-protein complexes formed in vitro were immunologically precipitated utilizing the beta-galactosidase moiety as a tagged antigen. The effectiveness of repeating cycles of immunoprecipitation was demonstrated by the color selection method designed for pUC-derived plasmids, after introducing the precipitated plasmids into Escherichia coli. After three cycles of DNA immunoprecipitation, only a few kinds of sequences constituted the majority. By repeating two more cycles, the most predominant sequence was finally enriched until homogeneous, indicating the enrichment of the binding sequences in a hierarchical order. Further application to human genomic DNA showed that two EcoRI DNA fragments, 0.49 and 0.78 kb in size, were exclusively identified. This procedure can be applied to the systematic analysis of binding sequences for any other DNA binding proteins without production of any specific antibodies or further purification.     

A procedure for the automatic determination of hydrophobic cores in protein structures.

An algorithm is described for automatically detecting hydrophobic cores in proteins of known structure. Three pieces of information are considered in order to achieve this goal. These are: secondary structure, side-chain accessibility, and side-chain-side-chain contacts. Residues are considered to contribute to a core when they occur in regular secondary structure and have buried side chains that form predominantly nonpolar contacts with one another. This paper describes the algorithm's application to families of proteins with conserved topologies but low sequence similarities. The aim of this investigation is to determine the efficacy of the algorithm as well as to study the extent to which similar cores are identified within a common topology.     

A procedure for urease and protein extraction from staphylococci

Staphylococcal cell protein and urease can be solubilized after growth in Todd-Hewitt broth supplemented with 0.5% yeast extract by extraction for 18-24 h in phosphate buffer, pH 7.0. In general 20% (but up to 100%) of the urease present in the original cells could be solubilized. Less protein was solubilized. Species examined included coagulase-negative staphylococci, Staphylococcus intermedius and Staph. aureus. Extracts of Staph. epidermidis prepared by this procedure gave electrophoretic urease and protein patterns similar to those prepared by sonication. The procedure was simple and minimized handling of the cells.     

A procedure for urease and protein extraction from staphylococci

Staphylococcal cell protein and urease can be solubilized after growth in Todd-Hewitt broth supplemented with 0.5% yeast extract by extraction for 18–24 h in phosphate buffer, pH 7.0. In general 20% (but up to 100%) of the urease present in the original cells could be solubilized. Less protein was solubilized. Species examined included coagulase-negative staphylococci, Staphylococcus intermedius and Staph. aureus. Extracts of Staph, epidermidis prepared by this procedure gave electrophoretic urease and protein patterns similar to those prepared by sonication. The procedure was simple and minimized handling of the cells.     

Combining ultracentrifugation and peptide termini group-specific immunoprecipitation for multiplex plasma protein analysis

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.     

Selective and efficient immunoprecipitation of the disease-associated form of the prion protein can be mediated by nonspecific interactions between monoclonal antibodies and scrapie-associated fibrils

Transmissible spongiform encephalopathies are characterized by the accumulation in brain tissues of an abnormal isoform of the prion protein named PrPsc, which is the only direct marker known for transmissible spongiform encephalopathies. Here we show that PrPsc can be specifically immunoprecipitated by using several monoclonal antibodies (mAbs) of various specificities independently of the properties of their binding site (paratope). These results strongly suggest that a significant proportion of mAbs can interact with PrPsc aggregates through nonspecific paratope-independent interactions allowing selective immunoprecipitation of PrPsc when these mAbs are immobilized on a polydisperse solid phase like microbeads.     

A new purification procedure for bovine C-reactive protein and serum amyloid P component.

1. A new purification procedure was started with salting-out fractionation of serum proteins at 45-75% saturated ammonium sulfate concentration, followed by HE agarose affinity chromatography by which calcium-dependently bound CRP and SAP were purely eluted with EDTA-containing buffer. 2. Pure CRP and SAP were finally separated by DEAE-5PW HPLC. 3. This procedure gave recovery of 15 and 26%, and fold purification of 2650 and 2400 for CRP and SAP, respectively. 4. Each subunit of CRP and SAP had one intrasubunit disulfide bond, determined by reduction and carboxymethylation.     

A novel procedure for protein extraction from formalin-fixed paraffin-embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.     

A rapid assay procedure for thiamine-binding protein of Escherichia coli

The advantages associated with the described immobilized enzyme reactor include: (1) Use of the common rate equations and kinetic parameters. (2) Detection of significant lag periods. (3) Quantitative measure for non-covalently attached enzyme. (4) The means for washing the immobilized enzyme allows for the repeated use of the same matrix-bound enzyme. (5) Constant temperature control. (6) Both unbound native and matrix-bound enzyme may be reacted under identical conditions. (7) No grinding of the glass-bound enzyme or other matrix fragmentation occurs because no abrasive forces are required for stirring.     

A DNase I binding/immunoprecipitation assay for actin

An actin assay which employs the competition between labeled and unlabeled rabbit skeletal muscle actin for DNase I has been developed. Iodination of actin by the method of Bolton and Hunter results in the incorporation of approximately 0.5 mol of 125-iodine/incorporation of approximately 0.5 mol of 125-iodine/mol of actin. This 125I-actin retained the ability to bind to DNase I and inhibit enzymatic activity. The 125I-actin-DNase complex can be precipitated by the addition of a monospecific rabbit antibody to DNase I. The efficiency of this immunoprecipitation step is improved by the use of a second sheep anti-rabbit gamma-globulin. Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound 125I-actin by rabbit skeletal muscle actin standards or by the actin present in tissue and cell extracts. Using 17.5 ng of DNase I and approximately 500 pg of 125I-actin, 50% inhibition of binding was obtained with 23 ng of unlabeled actin. Reducing the amount of DNase I to 2 ng results in 50% inhibition of binding with 4 ng of unlabeled actin and an increase in the estimated sensitivity of the assay from 1.7 to 0.24 ng. The slopes of the displacement curves generated with both vertebrate and invertebrate non-muscle actins are parallel to rabbit skeletal muscle actin. This observation indicates approximately equal actin-DNase I binding affinities and suggests a high degree of conservation of the actin-DNase I binding site. The assay is useful for measuring the pools of F- and G-actin in a wide range of cells.