ABSTRACT
Mitochondrial preparation is a key technique in the study of mitochondria. Growing evidence has demonstrated that mitochondrial proteins are tissue or cell type dependent. Locating the proteins in the global presence of mitochondrial membranes is a primary consideration in adopting antibodies for affinity enrichment of mitochondria on a micro scale. Two proteins located on the outer membrane of mitochondria, cytochrome b5 type B (CYB5B) and synaptojanin-2-binding protein (SYNJ2BP), were selected as candidates based on a survey of databases and the literature. The polyclonal antibodies against the truncated CYB5B and SYNJ2BP exhibited specific recognition to mitochondria and wider sensitivity to several tested mouse tissues and cell lines, whereas the antibody 22-kDa translocase of the outer mitochondrial membrane (TOM22) nearly missed detection of mitochondria in the liver and responded minimally to mitochondria from H9C2 and L-02 cells. Through the affinity enrichment for cellular mitochondria using magnetic beads coated with anti-CYB5B or anti-SYNJ2BP, we found that the anti-CYB5B beads could enrich mitochondria more efficiently even on a scale of 10,000 cultured cells. For the integrity and protein components, the enriched mitochondria on anti-CYB5B were carefully examined and were accepted in further functional study. We propose that an anti-CYB5B immunomagnetic approach is feasible in the micropreparation of mitochondria from cultured cells.
Subject(s)
Cell Fractionation/methods , Immunomagnetic Separation/methods , Mitochondria/chemistry , Mitochondria/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Animals , Antibody Affinity , Base Sequence , Cell Fractionation/standards , Cell Line , Cytochromes b5/genetics , Cytochromes b5/immunology , DNA Primers/genetics , Immunomagnetic Separation/standards , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Peptide Fragments/genetics , Peptide Fragments/immunology , Proteomics/methods , Quality Control , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
High background interference during the antibody pair screening process is inevitable. In this study, we found that the high background was associated with heterophilic antibody interference introduced by the application of ascites-derived monoclonal antibodies when conducting large-scale antibody pair screening against different proteins. To eliminate antibody-associated heterophilic antibody interference, both blocking with mouse normal sera and antigen-mediated affinity chromatography were used, resulting in significant improvement in pairing performance and in antibody pair screening efficiency.
Subject(s)
Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Artifacts , Immunoassay/methods , Animals , Cattle , Mice , Recombinant Proteins/immunologyABSTRACT
Two tyrosine residues (Tyr(4) and Tyr(76)) of succinyl-CoA:3-oxoacid CoA transferase (SCOT) are sensitive to nitric oxide (NO) stress, as assessed by mass spectrometry and site-direct mutagenesis. However, monitoring the SCOT nitration in tissue or cells is challenging. Herein, we describe the development of an assay to detect nitrated SCOT directly using site-specific antibodies; the monoclonal antibodies were generated and screened against nitrated peptides of SCOT. After stringent filtration, two antibodies, anti-SCOT4N and anti-SCOT76N, which specifically recognise Tyr(4) or Tyr(76) of SCOT, respectively, were successfully selected. In a cell model over-expressing iNOS in the mitochondria, nitrated SCOT was significantly increased compared with control cells. In addition, in a mouse model of diabetes, nitrated Tyr(4) and Tyr(76) in the heart and kidney were higher compared to the control animals. Our results using monoclonal antibodies against nitrated SCOT peptides are in good agreement with the proteomic data.
Subject(s)
Coenzyme A-Transferases/metabolism , Immunochemistry/methods , Immunohistochemistry/methods , Mitochondria/enzymology , Nitric Oxide/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Coenzyme A-Transferases/analysis , Diabetes Mellitus/metabolism , Female , Mice , Mice, Inbred Strains , Peptides/chemistry , Peptides/immunology , Peptides/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue DistributionABSTRACT
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ABSTRACT
Mouse model induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) is generally accepted as an ideal object to study on the carcinogenesis mechanisms of human colorectal cancer (CRC). The genomic responses to the AOM/DSS treatment in mouse that possibly lead to elucidation of CRC pathological mechanism are still poorly understood. For the first time, we investigated the cancer genome landscape of AOM/DSS mouse model by exome sequencing, to testify its molecular faithfulness to human CRC. Of 14 neoplastic samples, 7575 somatic variants were identified, which resulted in 2507 mutant genes and exhibited a large diversity in both colorectal aberrant crypt foci (ACF) and tumors even those tissues that were gained from the similar morphology or same treatment period. Cross-species comparison of the somatic variants demonstrated the totally different patterns of variable sites, mutant genes and perturbed pathways between mouse and human CRC. We therefore come to a conclusion that the tumorigenesis at genomic level in AOM/DSS model may not be properly comparable with that in human CRC, and the molecular mechanism elicited from this animal model should be carefully evaluated.
Subject(s)
Azoxymethane , Colorectal Neoplasms/chemically induced , Colorectal Neoplasms/genetics , Dextran Sulfate , Genetic Variation , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/genetics , Aberrant Crypt Foci/genetics , Animals , Humans , Male , Mice, Inbred C57BL/genetics , Mutation , Exome SequencingABSTRACT
In the genome of Thermoanaerobacter tengcongensis, three genes belonging to ROK (Repressor, ORF, and Kinase) family are annotated as glucokinases (GLKs). Using enzyme assays, the three GLKs were identified as ATP-dependent GLK (ATP-GLK), ADP-dependent GLK (ADP-GLK), and N-acetyl-glucosamine/mannosamine kinase (glu/man-NacK). The kinetic properties of the three GLKs such as K(m), V(max), optimal pH, and temperature were characterized, demonstrating that these enzymes performed the specific functions against varied substrates and under different temperatures. The abundance of ATP-GLK was attenuated when culture temperature was elevated and was almost undetectable at 80°C, whereas the ADP-GLK abundance was insensitive to temperature changes. Using degradation assays, ATP-GLK was found to have significantly faster degradation than ADP-GLK at 80°C. Co-immunoprecipitation results revealed that heat shock protein 60 (HSP60) could interact with ATP-GLK and ADP-GLK at 60 and 75°C, whereas at 80°C, the interaction was only effectively with ADP-GLK but not ATP-GLK. The functions of GLKs in T. tengcongensis are temperature dependent, likely regulated through interactions with HSP60.