Human EphB1 HEK293 Overexpression Lysate: Product Information
This Human EphB1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of EphB1 protein (Cat: 11963-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the human EPHB1 (P54762-1) extracellular domain (Met 1-Pro 540) was expressed, with a polyhistidine tag at the C-terminus.
The recombinant human EPHB1 consists of 534 amino acids and has a calculated molecular mass of 60 kDa as estimated in SDS-PAGE under reducing conditions.
Human EphB1 HEK293 Overexpression Lysate: Usage Guide
Cell lysate was prepared by homogenization of the over-expressed cells 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.
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.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human EphB1 HEK293 Overexpression Lysate: Alternative Names
Human ELK Overexpression Lysate; Human EPHT2 Overexpression Lysate; Human Hek6 Overexpression Lysate; Human NET Overexpression Lysate
EphB1 Background Information
Ephrin type-B receptor 1, also known as EphB1, belongs to the ephrin receptor subfamily of the protein-tyrosine kinase family which 16 known receptors (14 found in mammals) are involved: EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA1, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6. EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and that this phosphorylation event is crucial for syndecan-2 clustering and spine formation. The Eph family of receptor tyrosine kinases (comprising EphA and EphB receptors) has been implicated in synapse formation and the regulation of synaptic function and plasticity6. Ephrin receptors are components of cell signalling pathways involved in animal growth and development, forming the largest sub-family of receptor tyrosine kinases (RTKs). Ligand-mediated activation of Ephs induce various important downstream effects and Eph receptors have been studied for their potential roles in the development of cancer. EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB.
Dalva MB, et al. (2000) EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell. 103(6): 945-56.
Takasu MA, et al. (2002) Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors. Science. 295(5554): 491-5.
Adams RH, et al. (1999) Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 13(3): 295-306.
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