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|Human Cell lysate that Human KIRREL / NEPH1 / KIRREL1 transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|A DNA sequence encoding the human KIRREL (NP_060710.3) (Met1-Leu493) was expressed with the Fc region of human IgG1 at the C-terminus.|
|The recombinant human KIRREL/Fc comprises 715 amino acids and has a predicted molecular mass of 78.8 kDa. The apparent molecular mass of the protein is approximately 96.1 kDa in SDS-PAGE under reducing conditions.|
|Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
NEPH1 (KIRREL1) belongs to a family of three closely related transmembrane proteins of the Ig superfamily with a structure similar to that of nephrin. All three Neph proteins share a conserved podocin-binding motif; mutation of a centrally located tyrosine residue dramatically lowers the affinity of Neph1 for podocin. Neph1 triggers AP-1 activation similarly to nephrin but requires the presence of Tec family kinases for efficient transactivation. Neph1 consists of a signal peptide, five Ig-like C2-type domains with the middle domain overlapping with a PKD-like domain, an RGD sequence, a transmembrane domain and a cytoplasmic tail, which is expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. Neph1 is abundantly expressed in the kidney, specifically expressed in podocytes of kidney glomeruli, and plays a significant role in the normal development and function of the glomerular permeability. Neph1 interacts with nephrin in vitro and in vivo, and able to stimulate transcriptional activation in a model system, such as the activation the transcription factor AP-1 via the stimulation of a MAPK module. Neph1 is crucial for the integrity of the slit diaphragm, as Neph1 gene knockout mice results in effacement of glomerular podocytes, heavy proteinuria, and early postnatal death.