[Preparation of polyclonal antiserum against beta subunit of rabbit BK channel].
OBJECTIVE
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.


CONCLUSIONS
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.
Wang
Ya-rong
YR
Department of Radiology, Tangdu Hospital, Xi'an 710038, China. wangyar@fmmu.edu.cn
Wei
Jing-guo
JG
Sun
Oiang
O
Ma
Ke-jun
KJ
Zhang
Xiao-yong
XY
Znang
Yan-xia
YX
Han
Hua
H
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D007106:Immune Sera / Q000032:analysis / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007106:Immune Sera; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D011993:Recombinant Fusion Proteins