The sodium-dependent inorganic phosphate transporter SLC34A1 (NaPi-IIa) is not localized in the mouse brain: a case of tissue-specific antigenic cross-reactivity.

作者: Max Larsson , Cecilie Morland , Irais Poblete-Naredo , Jürg Biber , Niels Christian Danbolt

DOI: 10.1369/0022155411411713

关键词: Knockout mouseBiologyAntiserumImmunolabelingImmunofluorescenceNervous tissuePolyclonal antibodiesKidneyImmunohistochemistryMolecular biology

摘要: The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression nervous tissue. Western blots showed that immunoreactive band (~90 kDa) was only present wild-type kidney membranes not knockout or brain membranes. In water-soluble fraction of brains, another (~50 observed; this detected Light electron microscopic immunohistochemistry using antibodies immunolabeling tubules but mice. brain, labeling presynaptic nerve terminals also This pattern produced by preimmune serum. conclude specific toward kidney, caused cross-reaction with an unknown cytosolic protein tissue-specific cross-reactivity highlights potential pitfall when validating antibody specificity mouse-derived tissue other than interest underlines utility testing sera.

参考文章(12)
N.C. Danbolt, K.P. Lehre, Y. Dehnes, F.A. Chaudhry, L.M. Levy, LOCALIZATION OF TRANSPORTERS USING TRANSPORTER-SPECIFIC ANTIBODIES Methods in Enzymology. ,vol. 296, pp. 388- 407 ,(1998) , 10.1016/S0076-6879(98)96028-1
Setsuji Hisano, Hiromi Haga, Zhunan Li, Sawako Tatsumi, Ken-Ichi Miyamoto, Eiji Takeda, Yoshihiro Fukui, Immunohistochemical and RT-PCR detection of Na+-dependent inorganic phosphate cotransporter (NaPi-2) in rat brain Brain Research. ,vol. 772, pp. 149- 155 ,(1997) , 10.1016/S0006-8993(97)00884-6
Hugo Mouquet, Johannes F. Scheid, Markus J. Zoller, Michelle Krogsgaard, Rene G. Ott, Shetha Shukair, Maxim N. Artyomov, John Pietzsch, Mark Connors, Florencia Pereyra, Bruce D. Walker, David D. Ho, Patrick C. Wilson, Michael S. Seaman, Herman N. Eisen, Arup K. Chakraborty, Thomas J. Hope, Jeffrey V. Ravetch, Hedda Wardemann, Michel C. Nussenzweig, Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation Nature. ,vol. 467, pp. 591- 595 ,(2010) , 10.1038/NATURE09385
S. Holmseth, Y. Dehnes, L.P. Bjørnsen, J.-L. Boulland, D.N. Furness, D. Bergles, N.C. Danbolt, SPECIFICITY OF ANTIBODIES: UNEXPECTED CROSS-REACTIVITY OF ANTIBODIES DIRECTED AGAINST THE EXCITATORY AMINO ACID TRANSPORTER 3 (EAAT3) Neuroscience. ,vol. 136, pp. 649- 660 ,(2005) , 10.1016/J.NEUROSCIENCE.2005.07.022
Clifford B. Saper, Paul E. Sawchenko, Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry. The Journal of Comparative Neurology. ,vol. 465, pp. 161- 163 ,(2003) , 10.1002/CNE.10858
S. Holmseth, K. P. Lehre, N. C. Danbolt, Specificity controls for immunocytochemistry Anatomy and Embryology. ,vol. 211, pp. 257- 266 ,(2006) , 10.1007/S00429-005-0077-6
Linda H Bergersen, Jon Storm-Mathisen, Vidar Gundersen, Immunogold quantification of amino acids and proteins in complex subcellular compartments Nature Protocols. ,vol. 3, pp. 144- 152 ,(2008) , 10.1038/NPROT.2007.525
L. Beck, A. C. Karaplis, N. Amizuka, A. S. Hewson, H. Ozawa, H. S. Tenenhouse, Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities Proceedings of the National Academy of Sciences of the United States of America. ,vol. 95, pp. 5372- 5377 ,(1998) , 10.1073/PNAS.95.9.5372
S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, H. Murer, Expression cloning of human and rat renal cortex Na/Pi cotransport. Proceedings of the National Academy of Sciences of the United States of America. ,vol. 90, pp. 5979- 5983 ,(1993) , 10.1073/PNAS.90.13.5979
Heini Murer, Ian Forster, J�rg Biber, The sodium phosphate cotransporter family SLC34. Pflügers Archiv: European Journal of Physiology. ,vol. 447, pp. 763- 767 ,(2004) , 10.1007/S00424-003-1072-5