Human EED Baculovirus-Insect cells Overexpression Lysate

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Human EED Baculovirus-Insect cells Overexpression Lysate: Product Information

Product Description
This Human EED overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of EED protein (Cat: 11307-H20B) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect cells
Species
Human
Sequence Information
A DNA sequence encoding the human EED (O75530-1) (Met 1-Arg 441) was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
Molecule Mass
The recombinant human EED/GST chimera consists of 678 amino acids and has a calculated molecular mass of 78 kDa. It migrates as an approximately 75-85 kDa band in SDS-PAGE under reducing conditions.

Human EED Baculovirus-Insect cells Overexpression Lysate: Usage Guide

Preparation Method
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.
Lysis Buffer
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.
Recommend Usage
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.
Sample Buffer
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.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.

Human EED Baculovirus-Insect cells Overexpression Lysate: Alternative Names

Human HEED Overexpression Lysate; Human WAIT1 Overexpression Lysate

EED Background Information

EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes. EED depletion significantly impeded erythroid maturation. EED depletion impaired the generation of hematopoietic stem cells. The function of EED within polycomb repressive complex 2 (PRC2) is mediated by a complex network of protein-protein interactions. The targeted disruption of EED's reader function can lead to allosteric inhibition of PRC2 catalytic activity. Eed(Delta/Delta) HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other, and these conflicting events would lead to abnormal differentiation and functional defect of Eed(Delta/Delta) HSPCs.
Full Name
embryonic ectoderm development
References
  • Ura H, et al. (2011) Eed/Sox2 regulatory loop controls ES cell self-renewal through histone methylation and acetylation. EMBO J. 30(11): 2190-204.
  • Montgomery ND, et al. (2007) Molecular and functional mapping of EED motifs required for PRC2-dependent histone methylation. J Mol Biol. 374(5): 1145-57.
  • Jin Q, et al. (2003) The protein phosphatase-1 (PP1) regulator, nuclear inhibitor of PP1 (NIPP1), interacts with the polycomb group protein, embryonic ectoderm development (EED), and functions as a transcriptional repressor. J Biol Chem. 278(33): 30677-85.
  • Showell C, et al. (2002) Identification of putative interaction partners for the Xenopus Polycomb-group protein Xeed. Gene. 291(1-2): 95-104.
  • Rinchik EM, et al. (1993) N-ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse chromosome 7. Mamm Genome. 4(7): 349-53.
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