CARD12 Overexpression Lysate

Western Blot: CARD12 Overexpression Lysate (Adult Normal) [NBP2-06545] Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for CARD12.

• Reactivity: Hu
• Applications: WB

CARD12 Overexpression Lysate Summary

• Description:

  CARD12 Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC206757
  
  Accession#: NM_021209
  
  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: NLRC4

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: 116 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 CARD12 Overexpression Lysate

• CARD12leucine rich repeat and CARD domaincontaining 4
• caspase recruitment domain family, member 12
• Caspase recruitment domain-containing protein 12
• Clan protein
• CLAN1ipaf
• CLANCARD, LRR, and NACHT-containing protein
• CLR2.1
• Ice protease-activating factor
• Ipaf
• NLR family, CARD domain containing 4

Background

Ipaf (also known as Clan/CARD12) is a CARD domain containing protein. CARD (caspase-associated recruitment domain) proteins are key regulators of cell death, cell survival and cytokine production (reviewed in Damiano and Reed, 2004). In general CARD proteins are implicated in host defense against infection, environmental stress or cellular damage. CARD domains are found in the N-terminal pro-domains of certain caspases, a family of apoptotic and pro-inflammatory proteases, as well as in a diversity of other proteins including Ipaf/Clan/CARD12. CARD domains are homotypic protein interaction motifs that enable networks of proteins to communicate via CARD-CARD interactions. There are at least three major signaling pathways in which CARD proteins act: (1) Regulation of caspase activation in the context of apoptosis (2) Regulation of caspase activation in the context of inflammation (3) Regaultion of NF-kB activation in the context of innate or adaptive immune responses. As there is significant crosstalk between pathways that lead to caspase-mediated apoptosis or inflammation and pathways that result in NF-kB activation, it is logical that similar protein modules such as CARD domains are found repeatedly in proteins from all three pathways. Ipaf plays a role in regulating caspase-1 activity, which in turn mediates the maturation of inflammatory cytokines IL-1b and IL-18 (reviewed in Lu et al, 2005). In transfected cells, Ipaf has been shown to directly interact with procaspase-1 and induce proteolytic activation of procaspase-1 in transfected cells. On the flip side, macrophages from IPAF deficient mice failed to activate caspase-1 in response to Salmonella typhimurium infection underscoring the importance of IPAF in vivo. IPAF also interact with the pro-apoptotic adaptor protein ASC and co-expression of IPAF with ASC has been shown to induce NF-kB activation and 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: NLRC4