Calcium (Ca+2 or Ca) is the most abundant cation and it is required for many physiological activities such as bone formation and it acts as a second messenger in signal transduction. However only 1% of Ca is present in ionic form in biological fluids. Ca concentration is regulated by calcitropic hormones that act on bone, kidney, and intestine. Extracellular Ca+-levels are sensed and regulated by Calcium Sensing receptor (CASR). When Ca levels are limiting then it must be taken up by active, transcellular pathways comprising (1) Ca++ entry across apical membrane, (2) cytosolic transport of Ca++ across the cell form apical to basolateral membrane facilitated by a family of low mol wt Calcium binding proteins (CABPs) that include vitamin D3-dependent Ca++ binding proteins (calbindin-D9k, Calbindin-28k, Calretinin, Parvalbumin, S100, calmodulin) and finally (3) an active extrusion of Ca++ through basolateral membrane mediated by Ca++-ATPase and Na+-Ca++ exchangers (NCX). Ca++ absorption in intestine and its reabsorption in kidney are carried out by Ca++ Transport (CaT) proteins, CaT-1, CaT-2 or Epithelial Ca Channel (ECAC1/ECAC2/CaT-Like (CaT-L) proteins.

CAT1 (rat/mouse 727 aa; human 725 aa, chromosome 7q33-q34; ~85 kDa) is also known as ECAC2, CAT-like (CATL) or TRPV6 (transient receptor potential cation channel, subfamily V, member 6) contains a cytoplasmic N-terminus, 6 ankyrin repeat domains, 6 transmembrane domains, an extracellular pore region between TM5-6, and cytoplasmic C-terminus. CaT-1 shows 75% sequence identity to epithelial Ca++ channel (ECaC) protein from rabbit kidney. In rat, CaT-1 transcript (3-Kb) is expressed in duodenum, proximal jejunum cecum and colon but not in kidney; whereas ECaC in rabbit is expressed in apical cell membranes in distal part of nephron (kidney), duodenum, jejunum and placenta. The 6.5 Kb CaT-1-transcript in brain, thymus and adrenal gland and expressed sequences in rat and human brain and human prostate, spleen, placenta and lung suggest additional roles for CaT-1 beyond the intestinal Ca++ absorption.

CaT2 (rat 723 aa; human 729 aa, chromosome 7q35) is also known as ECAC1 or TRPV5) is ~75% homologous with CAT1. In contrast to CaT-1, CaT-2 is exclusively expressed in kidney and absent in intestine, brain, heart, liver, lung, skeletal muscle, spleen, thymus, testis, adrenal gland. It co-localizes with calbindin D28K and Na+ channel exchanger 1 (NCX1). CaT-2 mediates saturable apical Ca++ uptake by the cells of distal convoluted tubule and connecting segment of nephron where active re-absorption of Ca++ takes place via transcellular route. CaT-2 has moderate abilities to transport Sr++ and Ba++. A strong inhibition of CaT-2 mediated Ca++ transport by Cd++ leads to hypercalciurea and renal stone formation.

Calbindins are Ca-binding proteins belonging to the troponin C superfamily. CALB28K/Calbindin 1/CALB1 (D28K/Spot35 protein or cholecalcin, rat 261 aa; mouse 261 aa; human 261-aa, chromosome 8q21.3-q22.1) was originally described as 27-kDA induced by vitamin D in the duodenum of chicken. In mammals, it is expressed in the kidney, pancreatic islets, and brain. In brain, its synthesis is independent of vitamin D. CABP28K contains 4 active and 2 inactive EF-hand Ca-binding domains. The gene for CABP28K is clustered in the same region as carbonic anhydrase. The neurons in the brains of patients with Huntington disease are CAB28K depleted. There are two types of CaBPs: the "trigger"- and the "buffer"-CaBPs. The conformation of "trigger" type CaBPs changes upon Ca2+ binding and exposes regions on protein that interact with target molecules, thus altering their activity. The buffer-type CABP are thought to control the intracellular calcium concentration. Calbindin-D-28K is found predominantly in subpopulations of central and peripheral nervous system neurons, and in certain epithelial cells involved in Ca2+ transport such as distal tubular cells and cortical collecting tubules of the kidney, and in enteric neuroendocrine cells.

CABP9K (CALB3 or CABP1; mouse, rat, and human 79 aa; chromosome Xp; ~9 kDa) is a cytosolic Ca-binding protein initially found in rat pancreas. It is also expressed in intestine, placenta, uterus and kidney. Its expression is controlled by vitamin D and sex hormone in a tissue specific manner. In keeping with its role in Ca-transport, its expression is highest in duodenal villus enterocytes. It is further shown that CABP9K is only expressed in differentiated enterocytes. CABP9K gene also contains Cdx2-homeoprotein binding sites, and that Cdx2 may play a crucial role in CABP9K transcription.

Calretinin/CR/CLB2/CALB2/Calbindin D29K protein (mouse/rat/human 271 aa, chromosome 16q22.1, mol wt ~29 kDa) also belongs to the Calbindin family. It is most closely related to CABP28K (~55% identity). It may be alternatively spliced to a C-terminally truncated fragment, Calretinin-22K in some tumor cell lines. Calretinin is highly expressed in the cerebellum, olfactory bulb, and in auditory neurons. Calretinin gene inactivation in mice eliminated long-term potentiation induction in the gentate gyrus and impaired motor coordination.

S100 protein or Ca-binding protein A1/S100A1 is also a member of low mol wt, cytoplasmic, calcium binding troponin family. It is produced in the brain, muscle, and heart and a wide variety of normal and tumor cells. S100 has two subunits: S100-alpha (mouse/rat/human 94 aa, chromosome 1q21) and S100-beta (mouse, rat/human 92 aa chromosome 21q22.2-q22.3) that forms either homodimer (alpha-alpha known as S-100a(0) or beta-beta known as S-100b) and heterodimers (known as S-100a) of ~21 kDa. S100 alpha and beta chains show ~58% sequence identity. At least 9 different genes for S100 have been assigned on chromosome 21 namely S100A1-S1009. S-100 contains 2 EF-hand domains. S100A1 improves cardiac contractile performance both by regulating sarcoplasmic reticulum Ca ion handling myofbirilar Ca-ion responsiveness. Leakage of S100 protein by glial cells into the extracellualr matrix, CSF and serum are useful in monitoring traumatic brain injury.

Parvalbumin (PV or PVALB or oncomodulin) is a high affinity low mol wt (~12 kDa) cytoplasmic protein belonging to the troponin C superfamily. It is expressed in high levels only in fast-contracting muscles and at lower levels in brain and several endocrine tissues. PV has two isoform: alpha (rat/mouse/human 110-aa, 7p13-p11) beta form (mouse/rat/human 109-aa) shares approx. 48% identity (mouse PVs). The PV-alpha is less acidic (pI=5) than the Beta-isoform and has an additional amino acid in the C-terminal helix. PV-alpha is also expressed in inner ear. PV-beta isoform is expressed in many fetal tissues, placenta and tumors.

 

NT=near N-Terminus; CT=near C-Terminus; I=internal; CP=cytoplasmic; EC=Extracellular domains; H=human; M=mouse; R=rat, b=bovine; c=cat; f=frog, p=pig, gp=guinea pig

** Expected antibody crossreactivity information is mostly based upon high (>80%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera or antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.
Western blot +ve protein controls, where available, are semi-pure, pure or recombinant proteins that are formulated in SDS-PAGE sample buffer. They are recommended to be used for Western (load 10 ul/lane) for visualization with antibodies.