AtPCaP1 | Arabidopsis thaliana plasma membrane cation-binding protein-1
AS10 931 | clonality: polyclonal | host: rabbit | reactivity: A. thaliana, R. sativus, B.rapa, B.oleracea
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1 : 2000 with standard ECL (WB)
|Expected | apparent MW||
24.5 | 36 kDa
|Confirmed reactivity||Arabidopsis thaliana, Raphanus sativus (41 kDa), Brassica rapa (42 kDa), Brassica rapa var. glabra Regel (43 kDa), Brassica oleracea var. italica (41 kDa)|
|Not reactive in||
no confirmed exceptions from predicted reactivity known in the moment
It is confirmed that the nock-out mutant (SALK_022955, Col-0 background) has no mRNA or protein of PCaP1. In the immunoblot, crude membrane fractions prepared from wild-type and pcap1 knock-out mutant plants were examined. Therefore the antibody to PCaP1 specifically recognizes PCaP1 and can be used for immunoblotting. For application in immunocytochemistry cross reacting band in proximity of Rubisco has to be removed by incubation of an antibody with a part of memrane with background band.
(1) Homogenize roots or root tips with the buffer without SDS.
(2) Centrifuge the homogenate in Eppendorf tubes at 2 000 rpm for 2 min to remove cell wall, nuclei, plastids and mitochondria.
(3) Centrifuge the obtained supernatant at 30 000 g for 20 min to separate soluble components. Please use adequate tubes for ultracentrifugation.
(4) Suspend the obtained precipitate (crude membranes) with SDS buffer with DTT or beta-mercaptoethanol.
(5) Heat the sample solution obtained the step (4) at 70 C for 5 min.
(6) Apply the denatured sample to SDS-PAGE.
Courtesy Prof. Msayoshi Maeshima, Nagoya University, Japan
|Selected references||Ide et al. (2007) Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane. J. Exp. Botany, 58: 1173-1183.
Nagasaki et al. (2008) A hydrophilic cation-binding protein of Arabidopsis thaliana, AtPCaP1, is localized to plasma membrane via N-myristoylation and interacts with calmodulin and the posphatidylinositol phosphates PtdIns(3,4,5)P3 and PtdIns(3,5)P2. FEBS J. 275: 2267-2282.
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