Subject(s)
Neuroprotective Agents , Parkinson Disease , Proanthocyanidins , Apoptosis/drug effects , Autophagy/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Neuroprotective Agents/pharmacology , Parkinson Disease/drug therapy , Proanthocyanidins/pharmacology , Reactive Oxygen Species , Rotenone/toxicityABSTRACT
AIM: To prepare and characterize the polyclonal antibody against human VSTM1. METHODS: VSTM1 has two main isoforms, VSTM1-v1, a type I transmembrane protein, and VSTM1-v2, a classical secretory protein, lacking only the transmembrane domain compared with VSTM1-v1. Two recombinant prokaryotic proteins of VSTM1-v2, Trx-His-S-VSTM1-v2 and GST-VSTM1-v2, were constructed, expressed, purified, and then used for immunization of New Zealand rabbits to prepare anti-VSTM1 antibody and coupling with CNBr-activated Sepharose 4B to purify the antibody by immunoaffinity chromatography, respectively. ELISA was performed to detect the titers of the antiserums. After purification, the antibody was identified by Western blotting and immunofluorescence cytochemistry. RESULTS: The titers of the antiserums from the two immunized rabbits were both 1:10(6);. Western blotting confirmed that the purified antibody could recognize both the overexpressed and endogenous VSTM1 specifically. Immunofluorescence cytochemistry verified that the antibody could also recognize the overexpressed VSTM1-v1 on the surface of HEK293T cells. CONCLUSION: The rabbit antibody against human VSTM1 of a high titer has been obtained, which can be used for recognizing endogenous VSTM1 in immunofluorescence cytochemistry.
Subject(s)
Antibodies/analysis , Receptors, Immunologic/analysis , Animals , Antibodies/immunology , Antibodies/isolation & purification , Antibody Specificity , Cloning, Molecular , HEK293 Cells , Humans , Immunization , Male , Rabbits , Receptors, Immunologic/genetics , Receptors, Immunologic/immunologyABSTRACT
AIM: To prepare, purify and characterize the polyclonal antibodies against CMTM4. METHODS: Four polypeptides named peptide 1, 2, 3, and 4 were synthesized based on the bioinformatics analysis of the three isoforms of CMTM4, CMTM4-v1, -v2, and -v3, and coupled with keyhole limpet hemocyanin (KLH) for immunization. Among them, peptide 1, 2, and 3, common for CMTM4-v1, v2, and v3, were mixed for immunization to prepare the antibody which can recognize all of the three isoforms (Ab1); while peptide 4, which is specific for CMTM4-v1, was injected separately into New Zealand rabbits to prepare the antibody specifically targeting CMTM4-v1 (Ab2). ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, Ab1 and Ab2 were identified by Western blot and immunohistochemistry assays. RESULTS: The titers of Ab1 and Ab2 were 1:10(5) and 1:10(6), respectively. Western blot confirmed their high specificity. Ab1 could also be used for immunohistochemistry analysis, but Ab2 could not. Immunohistochemistry analysis with Ab1 demonstrated the high expression level of CMTM4 in human testis, which was consistent with the previous result of Northern blot. Moreover, Western blot and immunohistochemistry analysis verified that Ab1 can also recognize mouse Cmtm4. CONCLUSION: Two specific antibodies against human CMTM4 were obtained, which will be helpful for further study.
