Prokaryotic expression, polyclonal antibody preparation, and sub-cellular localization analysis of Na+, K+-ATPase beta2 subunit.
Na+, K+-ATPase beta2 subunit (NKA1b2) is not only a regulator of Na+, K+-ATPase, but also functions in the interaction between neuron and glia cells as a Ca2+-dependent adhesion molecule. To further study the function of NKA1b2, the anti-NKA1b2 polyclonal antibody was prepared to recognize the outer-membrane carboxyl portion segment of NKA1b2. The coding region for amino acids 190-290 at the carboxyl portion of NKA1b2 (NKA1b2-CP) was sub-cloned into the vector pGEX-4T-2 and introduced into the Escherichia coli BL21(DE3) cell for efficient soluble expression. The amino acid sequence of expressed protein was determined using mass spectrometry following Mascot analysis. After purification, GST-NKA-beta2-CP was used to immunize the adult rabbits following standard protocols. The produced antiserum could detect the NKA1b2 protein expressed not only in the prokaryotic cells (E. coli) but also in the eukaryotic cells (COS7) transfected with NKA1b2 expression vector (pEGFP-NKA1b2). Furthermore, the antiserum was used for determining the localization of NKA1b2 in primary culture of neonatal rat neurons using immunohistochemical technique. Results demonstrated that NKA1b2 was localized both in the cytoplasm and cellular membrane. The preparation of anti-NKA-beta2-CP polyclonal antibody will facilitate further functional study on NKA1b2.
Beijing Institute of Radiation Medicine, Beijing 100850, PR China.
Ting-Fang
TF
Chen
Yong-Liang
YL
Zhang
Wen-Lin
WL
Xu
Zhong-Qiu
ZQ
Li
Bing
B
Hou
Chun-Li
CL
Wang
Ming
M
Fan
Ling-Jia
LJ
Qian
Ren-Ping
RP
Zhou
Cheng-Gang
CG
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011387:Prokaryotic Cells / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016415:Sequence Alignment; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; C503199:Na K ATPase beta2 subunit, rat; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D000254:Sodium-Potassium-Exchanging ATPase