We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 x anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.
Animals ; Antibodies, Bispecific/*immunology ; HEK293 Cells ; Haptens/*immunology ; Humans ; Immunoblotting/*methods ; Immunoprecipitation/methods ; Rabbits ; Single-Chain Antibodies/immunology

OBJECTIVETo investigate the optimum proportion of Mosaicplasty and genes-enhanced tissue engineering for the repair of acute osteochondral defects.

METHODSWestern blot test was conducted to detect the expression of hTGF-beta1, Col II and Aggrecan in 3 groups, including hTGF-beta1, transduction group, Adv-betagal transduction group and control group without transduction. Eighteen 6-month-old Shanghai male goats (weight: 22 to 25 kg) were used. BMSCs were isolated from the autologous bone marrow, and were subcultured to get the cells at passage 3. Thirty-six medial femoral condyles were used and divided into 6 groups named AR, AL, BR, BL, CR, and CL. Acute cylindrical defects (9 mm in diameter and 3 mm in depth)were created in the weight bearing area of the medial femoral condyle of hind limbs. In the single group, the autologous osteochondral mosaicplasty was performed to repair the defect; in the combination group, besides the mosaicplasty, the dead space between the cylindrical grafts and the host cartilage were injected with the suspension of hTGF-beta1, gene enhanced autogenous BMSCs in sodium alginate, and CaCl2 was dropped into it to form calcium alginate gels. The autologous osteochondral transplantation cover rates of group AR was 44.44% single group, AL was 44.44% combination group, BR was 33.33% single group, BL was 33.33% combination group, CR was 22.22% single group, and CL was 22.22% combination group. The goats were killed 24 weeks after operation to receive gross and histology observation, which was evaluated by the histological grading scale of O'Driscoll, Keeley and Salter. Immunohistochemistry and TEM observation were also performed.

RESULTSWestern blot test showed the expression of the hTGF-beta1, Col II and the Aggrecan in the hTGF-beta1 transduction group were significantly higher than that of the Adv-betaga1 transduction and the blank control groups. The gross and histology observation revealed that each defects of six groups had different degrees of repairing. There was no significantly difference among the BL, AR, and AL groups. But the scores of the other three groups (BR, CR, and CL) were significantly poorer than the former three groups.

CONCLUSIONMosaicplasty associated with genes enhanced tissue engineering could repair the osteochondral defects effectively. With the autologous osteochondral transplantation coverage reducing, the advantage of the combination could have a better representation.

Animals ; Blotting, Western ; Bone Diseases ; metabolism ; pathology ; therapy ; Cell Line ; Goats ; Humans ; Immunoprecipitation ; Male ; Tissue Engineering ; methods

OBJECTIVEMachado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 (CK2).

METHODSThe interaction between ataxin-3 and CK2 was identified by glutathione S-transferase (GST) pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results (1) Both wild type and expanded ataxin-3 interacted with CK2alpha and CK2beta in vitro. (2) In 293 cells, both wild type and expanded ataxin-3 interacted with CK2beta, but not CK2alpha. (3) CK2 phosphorylated wild type and expanded ataxin-3.

CONCLUSIONAtaxin-3 is a substrate of protein kinase CK2.

Ataxin-3 ; Casein Kinase II ; metabolism ; Cell Line, Transformed ; Glutathione Transferase ; metabolism ; Humans ; Immunoprecipitation ; methods ; Nerve Tissue Proteins ; metabolism ; Nuclear Proteins ; metabolism ; Phosphorylation ; Repressor Proteins ; metabolism ; Transfection ; methods

OBJECTIVETo develop a real-time PCR-based chromatin immunoprecipitation (ChIP) assay for determining the effect of sodium butyrate on acetylation of histone in gamma-globin gene promoter regions in K562 cells.

METHODSK562 cells were grown in the presence or absence of 0.5 mmol/L sodium butyrate for 48 h, and 1=10(7) cells per group were used for real-time PCR-based ChIP with anti-acetylated histone H3 or H4 antibodies. The levels of acetylated histone H3 and H4 (acH3 and acH4) in Ggamma- and Agamma-globin gene promoter regions were measured.

RESULTSIn the K562 cells with sodium butyrate treatment or without any treatment, the levels of acH3 or acH4 in Ggamma- or Agamma-globin gene promoter were higher than that in the necdin gene (negative control). Compared with the untreated K562 cells, the cells treated with 0.5 mmol/L sodium butyrate showed a 3.1-fold or 2.6-fold increase in acH3 or acH4 in Ggamma-globin gene promoter region, with also a 3.7-fold or 3.2-fold increase in acH3 or acH4 in Agamma-globin gene promoter region, respectively (P<0.01).

CONCLUSIONWe have successfully developed a real-time PCR-based ChIP assay for analyzing the acetylation of histone H3 and H4 in gamma-globin gene promoter regions. Our results support the role of sodium butyrate in increasing the level of acetylated histone in gamma-globin gene promoter regions.

Acetylation ; Butyrates ; pharmacology ; Chromatin Immunoprecipitation ; methods ; Histones ; chemistry ; Humans ; K562 Cells ; Promoter Regions, Genetic ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; gamma-Globins ; genetics

Hepatitis B virus (HBV) is a major cause of chronic liver disease, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. HBV polymerase (Pol) is an essential viral protein that is important for HBV replication and might be involved in the development of hepatocellular carcinoma. Protein-protein interactions appears to be crucial for its role. The aim of this study was to screen and identify the proteins that interact with Pol using a co-immunoprecipitation-based LC-MS/MS identification technique. The HBV Pol gene was amplified by polymerase chain reaction (PCR) and cloned into pCDNA3.1(+). The recombinant plasmid pCDNA3. 1(+)-Pol-flag was transfected into HeLa cells. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 45 proteins that co-immunoprecipitated with flag-tagged HBV Pol. Eleven of these have previously been reported as proteins that interact with HBV Pol. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 45 identified proteins and HBV Pol. Among these proteins, four proteins may play a role in three major molecular cellular networks, and are therefore worthy of further investigation.
Cell Line, Tumor ; Chromatography, Liquid ; methods ; Gene Products, pol ; chemistry ; genetics ; metabolism ; Hepatitis B ; genetics ; metabolism ; virology ; Hepatitis B virus ; chemistry ; enzymology ; genetics ; Humans ; Immunoprecipitation ; methods ; Protein Interaction Maps ; Software ; Tandem Mass Spectrometry ; methods

To detect the phosphorylation of potassium channel protein (Kv1.2 and Kv1.5) in rat cardiac muscle cells accurately, we applied the combined method of immunoprecipitation and Western blot in this study. Compared with using Western blot alone, the combination of immunoprecipitation and Western blot displayed high sensitivity to detect the activation of potassium channel proteins. Because of its simplicity, quickness and reproducibility, we find that this method was promising for detecting the phosphorylation of Kv1.2 and Kv1.5 proteins or other potassium channel proteins in rat cardiac muscle cells.
Animals ; Blotting, Western ; methods ; Cell Membrane ; metabolism ; Female ; Immunoprecipitation ; methods ; Kv1.2 Potassium Channel ; analysis ; genetics ; metabolism ; Kv1.5 Potassium Channel ; analysis ; genetics ; metabolism ; Male ; Myocytes, Cardiac ; cytology ; metabolism ; Phosphorylation ; Rats ; Rats, Sprague-Dawley

PURPOSE: The purpose of the present study was to investigate the aberrance of histone H3 lysine 4 trimethylation (H3K4me3) in patients with IgA Nephropathy (IgAN). MATERIALS AND METHODS: In this study, H3K4me3 variations in peripheral blood mononuclear cells (PBMCs) from 15 IgAN patients and 15 healthy subjects were analyzed using chromatin immunoprecipitation linked to microarrays analysis (ChIP-chip). ChIP real-time PCR was used to validate the microarray results. Expression analysis by quantitative real-time PCR (qRT-PCR) revealed correlations between mRNA and H3K4me3 levels. DNA methylation status was analyzed by quantitative methylation-specific PCR. RESULTS: We found that 321 probes displayed significant H3K4me3 differences in IgAN patients compared with healthy controls. Among these probes, 154 probes displayed increased H3K4me3 and 167 probes demonstrated decreased H3K4me3. For further validation, we selected 4 key relevant genes (FCRL4, GALK2, PTPRN2 and IL1RAPL1) to study. The results of ChIP real-time PCR coincided well with the microarray data. Quantitative RT-PCR revealed the correlations between the mRNA expression and the methylation levels of H3K4me3. Different degrees of DNA methylation alterations appeared on the selected positive genes. CONCLUSION: Our studies indicated that there were significant alterations in H3K4me3 in IgAN patients. These findings may help to explain the disturbed immunity and abnormal glycosylation involved in IgAN patients.
Adult ; Case-Control Studies ; Chromatin Immunoprecipitation ; Female ; Glomerulonephritis, IGA/*genetics/*metabolism ; Histones/*metabolism ; Humans ; Leukocytes, Mononuclear/*metabolism ; Lysine/*metabolism ; Male ; Methylation ; Oligonucleotide Array Sequence Analysis/*methods ; Real-Time Polymerase Chain Reaction ; Young Adult

Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.
Binding Sites ; Cell Line ; Heterogeneous-Nuclear Ribonucleoproteins ; isolation & purification ; metabolism ; Humans ; Immunoprecipitation ; methods ; Polypyrimidine Tract-Binding Protein ; isolation & purification ; metabolism ; RNA ; isolation & purification ; metabolism ; RNA-Binding Proteins ; isolation & purification ; metabolism

A marine unicellular green alga, Platymonas subcordiformis, was demonstrated to photobiologically produce hydrogen gas from seawater. The objective of this study was to localize and identify the hydrogenase isolated from P. subcordiformis. Adaptation in the presence of inhibitors of protein biosynthesis indicated that the hydrogenase was much more inhibited by cycloheximide than that by chloramphenicol. The result suggested that the hydrogenase isolated from P. subcordiformis is probably synthesized in cytoplasmic ribosomes. Both Western blot analysis and immunogold electron microscopy demonstrate that the P. subcordiformis hydrogenase is mainly located in the chloroplast stroma. The proteins that reacted specifically with the antibodies against the iron hydrogenase isolated from Chlamydomonas reinhardtii were concentrated by immunoprecipitation. The separated protein bands were cut out of the SDS-PAGE gel, in-gel digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Mascot was employed for analysis of the MALDI data using the public databases NCBInr. The hydrogenase isolated from P. subcordiformis was identified to be the Fe-hydrogenase.
Algal Proteins ; isolation & purification ; metabolism ; Biocatalysis ; drug effects ; Blotting, Western ; Chloramphenicol ; pharmacology ; Chlorophyta ; enzymology ; Cycloheximide ; pharmacology ; Cytoplasm ; enzymology ; ultrastructure ; Electrophoresis, Polyacrylamide Gel ; Hydrogenase ; antagonists & inhibitors ; isolation & purification ; metabolism ; Immunoprecipitation ; methods ; Iron-Sulfur Proteins ; antagonists & inhibitors ; isolation & purification ; metabolism ; Kinetics ; Microscopy, Immunoelectron ; Protein Synthesis Inhibitors ; pharmacology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

In the post-genomic era, various computational methods that predict protein-protein interactions at the genome level are available; however, each method has its own advantages and disadvantages, resulting in false predictions. Here we developed a unique integrated approach to identify interacting partner(s) of Semaphorin 5A (SEMA5A), beginning with seven proteins sharing similar ligand interacting residues as putative binding partners. The methods include Dwyer and Root-Bernstein/Dillon theories of protein evolution, hydropathic complementarity of protein structure, pattern of protein functions among molecules, information on domain-domain interactions, co-expression of genes and protein evolution. Among the set of seven proteins selected as putative SEMA5A interacting partners, we found the functions of Plexin B3 and Neuropilin-2 to be associated with SEMA5A. We modeled the semaphorin domain structure of Plexin B3 and found that it shares similarity with SEMA5A. Moreover, a virtual expression database search and RT-PCR analysis showed co-expression of SEMA5A and Plexin B3 and these proteins were found to have co-evolved. In addition, we confirmed the interaction of SEMA5A with Plexin B3 in co-immunoprecipitation studies. Overall, these studies demonstrate that an integrated method of prediction can be used at the genome level for discovering many unknown protein binding partners with known ligand binding domains.
Binding Sites ; genetics ; Cell Line, Tumor ; Cluster Analysis ; Computational Biology ; methods ; Databases, Protein ; Gene Expression Profiling ; Humans ; Hydrophobic and Hydrophilic Interactions ; Immunoprecipitation ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; Nerve Tissue Proteins ; chemistry ; genetics ; metabolism ; Neural Cell Adhesion Molecules ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Interaction Mapping ; methods ; Protein Structure, Tertiary ; Reverse Transcriptase Polymerase Chain Reaction