Human SEMA4A HEK293 Overexpression Lysate: Product Information
This Human SEMA4A overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of SEMA4A protein (Cat: 11756-H02H) from the overexpression lysate was verified.
A DNA sequence encoding the human SEMA4A (NP_071762.2) extracellular domain (Met 1-His 683) was fused with the Fc region of human IgG1 at the C-terminus.
The secreted recombinant human SEMA4A/Fc chimera is a disulfide-linked homodimer. The reduced monomer consists of 893 amino acids and predictes a molecular mass of 99 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rh SEMA4A/Fc monomer is approximately 110 kDa due to glycosylation.
Human SEMA4A 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 SEMA4A HEK293 Overexpression Lysate: Alternative Names
Human CORD10 Overexpression Lysate; Human RP35 Overexpression Lysate; Human SEMAB Overexpression Lysate; Human SEMB Overexpression Lysate
SEMA4A Background Information
Semaphorin-4A, also known as Semaphorin-B, SEMA4A, Sema B and SEMAB, is a single-pass type I membrane protein which belongs to thesemaphorin family. It inhibits axonal extension by providing local signals to specify territories inaccessible for growing axons. Semaphorin-4A / SEMA4A contains oneIg-like C2-type (immunoglobulin-like) domain, onePSI domain and oneSema domain. Defects in SEMA4A are the cause of retinitis pigmentosa type 35 (RP35) which leads to degeneration of retinal photoreceptor cells. Patients typically have night vision blindness and loss of midperipheral visual field. As their condition progresses, they lose their far peripheral visual field and eventually central vision as well. Defects in SEMA4A are also the cause of cone-rod dystrophy type 1 (CORD1) which are inherited retinal dystrophies belonging to the group of pigmentary retinopathies. CORDs are characterized by retinal pigment deposits visible on fundus examination, predominantly in the macular region, and initial loss of cone photoreceptors followed by rod degeneration. Semaphorins are secreted, transmembrane, and GPI-linked proteins, defined by cysteine-rich semaphorin protein domains, that have important roles in a variety of tissues. Humans have 2 semaphorins, Drosophila has five, and two are known from DNA viruses. Semaphorins are found in nematodes and crustaceans but not in non-animals. They are grouped into eight classes on the basis of phylogenetic tree analyses and the presence of additional protein motifs. Semaphorins have been implicated in diverse developmental processes such as axon guidance during nervous system development and regulation of cell migration.
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