BFAR Overexpression Lysate

Western Blot: BFAR Overexpression Lysate (Adult Normal) [NBP2-04359] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for BFAR.

• Reactivity: Hu
• Applications: WB

BFAR Overexpression Lysate Summary

• Description:

  BFAR Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC200078
  
  Accession#: NM_016561
  
  Protein Tag: C-MYC/DDK
  
  You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.
• Gene: BFAR

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.
  
  Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.
• Theoretical MW: 52.6 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at -80C. Avoid freeze-thaw cycles.
• Buffer: RIPA buffer

Lysate Details for Array

• Type: Overexpression

Notes

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Alternate Names for BFAR Overexpression Lysate

• BARRNF47RING finger protein 47
• bifunctional apoptosis inhibitor
• bifunctional apoptosis regulator

Background

BAR (bifunctional apoptosis regulator) is a multidomain protein that was originally identified as an inhibitor of Bax-induced apoptosis (Zhang et al, 2000). Apoptosis induction can be divided up into two major pathways, extrinsic and intrinsic. The extrinsic pathway is represented by death receptor signaling and the intrinsic pathway depends on mitochondrial events. BAR is in anchored in intracellular membranes and is thought to be a scaffold protein that may bridge components of both extrinsic and intrinsic apoptosis pathways through its antiapoptotic domains: 1. BAR contains a DED (death effector domain)-like protein interaction domain that suppresses death receptor apoptosis signaling pathways. Death receptors such as the TNF (tumor necrosis factor)-family contain protein interaction domains called DD (death domains) in their cytosolic regions. DD-containing TNF receptor family members such as Fas aggregate upon binding ligand and bind to an adaptor protein FADD which contains both DD and DED domains. The Fas/FADD complexes bind to the caspase family members such as 8 and 10 which contain DEDs in their N-terminal prodomain. This is followed by proteolytic processing and caspase activation, thereby initiating a signal transduction cascade leading to activation of downstream effector caspases, substrate cleavage, and ultimate cell death. DED-containing antiapoptotic proteins like BAR function as transdominant apoptosis inhibitors by competing for binding to the DED domains of proapoptotic proteins like Fadd, caspase-8 and caspase-10, thereby preventing assembly of functional death-inducing complexes and hence activation of downstream apoptosis signaling cascades. 2. BAR also contains a domain that mediates interactions with Bcl-2 family proteins and that is required for suppression of Bax-induced cell death in yeast and mammalian cells. Although the physiological functions of BAR remain to be elucidated. BAR is highly expressed in the brain and expression patterns as well as functional data with neuronal cell lines suggest that BAR is involved in regulating neuronal survival (Roth et al. 2003). Additionally, subcellular localization studies indicate that BAR predominantly localizes to the endoplasmic reticulum (ER), irrespective of cell type. Bcl-2 family proteins also localize to the ER. There is important crosstalk between the ER and mitochondria in the execution of cell death. It is thought that both BAR and Bcl-2 proteins play a role in regulating cell death/apoptosis induced by ER stress. Dysregulation of ER homeostasis and apoptosis is thought to be involved in the pathogenesis of some human neuronal diseases, including Alzheimer's, Parkinson's, polyglutamine diseases, nueronal storage diseases, prion dieases, as well as acute neurodegeration from brain trauma (reviewed in Lindholm et al, 2006). Since BAR is normally widely expressed in the brain, it may have a cytoprotective function in helping neurons to survive for the entire lifetime of the organism by playing a central role in inhibiting ER initiated apoptosis.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Bioinformatics

• Gene Symbol: BFAR