Human Semaphorin 5A HEK293 Overexpression Lysate: Product Information
This Human Semaphorin 5A overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Semaphorin 5A protein (Cat: 11300-H02H) from the overexpression lysate was verified.
A DNA sequence encoding the amino acids (Met 1-Thr 765) of human SEMA5A (NP_003957.2) extracellular domain was fused with Fc region of human IgG1 at the C-terminus.
The recombinant human SEMA5A (aa 1-765)/Fc is a disulfide-linked homodimer. The reduced monomer consists of 984 amino acids and predictes a molecular mass of 110.7 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rh SEMA5A/Fc monomer is approximately 125-135 kDa due to glycosylation.
Human Semaphorin 5A 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 Semaphorin 5A HEK293 Overexpression Lysate: Synonyms
Human SEMAF Overexpression Lysate; Human semF Overexpression Lysate
Semaphorin 5A Background Information
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. Semaphorin-5A, also known as Semaphorin-F, Sema F, SEMA5A and SEMAF, is a single-pass type I membrane protein that belongs to the semaphorin family. Semaphorin5A / SEMA5A contains one PSI domain, one Sema domain and seven TSP type-1 domains. It may act as positive axonal guidance cues. Semaphorin5A / SEMA5A is an axon regulator molecule and plays major roles during neuronal and vascular development. It plays an essential role in embryonic development. Semaphorin5A / SEMA5A induces endothelial cell migration from pre-existing vessels. It also plays a role in autism, reducing the ability of neurons to form connections with other neurons in certain brain regions.
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