APAF-1 Overexpression Lysate

Western Blot: APAF1 Overexpression Lysate (Adult Normal) [NBP2-04870] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for APAF1.

• Reactivity: Hu
• Applications: WB

APAF-1 Overexpression Lysate Summary

• Description:

  APAF-1 Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC213433
  
  Accession#: NM_181869
  
  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: APAF1

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: 37.8 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 APAF-1 Overexpression Lysate

• APAF1
• APAF-1
• APAF-1apoptotic protease activating factor 1
• apoptotic peptidase activating factor 1
• apoptotic peptidase activating factor
• apoptotic protease activating factor
• apoptotic protease-activating factor 1
• CED4
• DKFZp781B1145
• KIAA0413

Background

Apaf 1 (apoptosis protease-activating factor-1) is a key regulator of the mitochondrial apoptotic pathway (reviewed in Campioni et al, 2005), being the central element of the multimeric apoptosome. The apoptosome consists of cytochrome c, procaspase-9 and seven Apaf 1 monomers, and is considered to be core apoptotic machinery that executes mitochondria-dependent apoptosis. Cytochrome c, normally compartmentalized in the mitochorndria, is released into the cytoplasm following apoptotic stimuli. Apaf 1 binds cytochrome c in the cytoplasm and in the presence of dATP/ATP forms the apoptosome. The apoptosome binds procaspase-9 and promotes its autocatalytic activation. Active caspase-9, in turn, activates downstream caspases including 3, 6, and 7 contributing to the proteolytic caspase activation cascade which leads to cell death. Apaf 1 is an approx. 130 kDa protein. Alternative Apaf 1 splicing isoforms of Apaf 1 have been identified in mammalian cells, each having either or both an 11 amino acid insertion in the N-terminus and an additional WD40 repeat (43 aa) in the C-terminus; please refer to AceView of NCBI (http://www.ncbi.nih.gov/IEB/Research/Acembly/) for detailed information. Additionally, Apaf 1 can be proteolytically processed by active caspase-3 during apoptosis resulting in an Apaf 1 cleavage fragment of ~84 kDa (Lauber et al. 2001).

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: APAF1