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Published on: June 29, 2016
[Preparation of polyclonal antiserum against beta subunit of rabbit BK channel]
Ya-rong Wang1, Jing-guo Wei, Oiang Sun
1Department of Radiology, Tangdu Hospital, Xi'an 710038, China. wangyar@fmmu.edu.cn
Aim:
To prepare polyclonal antiserum against beta subunit of rabbit BK channel in mice.
Methods:
Gene encoding the intracellular fragment of rabbit BK channel's beta subunit was amplified by RT-PCR. The GST-beta fusion protein was expressed in E. coli. The fusion protein from PAGE gel was used to immunize BALB/c mice and prepare polyclonal antiserum. The specificity of antiserum was identified by ELISA and Western blot.
Results:
A unique band about 300 bp was amplified by RT-PCR and was verified to be BK channel beta subunit by DNA sequencing. The SDS-PAGE analysis showed that the M(r) of the fusion protein was about 37,000. The purity of GST-beta fusion protein was over 95%. The polyclonal antiserum against GST-beta fusion protein could recognize both GST-beta fusion protein and beta protein in rabbit tissues. The highest titer of the antiserum was about 1:128,000, as shown by Western blot and ELISA, respectively.
Conclusion:
The gene encoding the intracellular fragment of rabbit BK channel's beta subunit has been cloned. The polyclonal antiserum against beta subunit of rabbit BK channel with high titer and specificity has been prepared successfully.
