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Human KEAP1 / INRF2 Insect Cell Lysate (WB positive control)

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Human KEAP1 Transfected / Overexpression Cell Lysate Product Information
Expressed Host:Baculovirus-Insect cells
Product Description:Baculovirus-Insect Cell lysate that Human KEAP1 / INRF2 transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).
Sequence information:A DNA sequence encoding the human KEAP1 (Q14145) (Gln 2-Cys 624) was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus linked by the AVI tag.
Predicted N Terminal:Met
Molecule Mass:The recombinant human KEAP1/GST chimera consists of 875 amino acids and has a calculated molecular mass of 99.2 kDa. It migrates as an approximately 85 kDa band in SDS-PAGE under reducing conditions.
Human KEAP1 Transfected / Overexpression Cell Lysate Usage Guide
Preparation Method:Cell lysate was prepared by homogenization 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.
Quality Control Testing:12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.
Stability:Samples are stable for up to twelve months from date of receipt.
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. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.
Storage Buffer:1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Storage Instruction:Store at 4℃. After re-dissolution, aliquot and store at -80℃.
Application notes:Western blot (WB): Use at an assay dependent dilution.
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
KEAP1 Background

Kelch-like ECH-associated protein 1, also known as cytosolic inhibitor of Nrf2, Kelch-like protein 19, KEAP1 and INRF2, is a cytoplasm and nucleus protein which contains one BACK (BTB/Kelch associated) domain, one BTB (POZ) domain and six Kelch repeats. KEAP1 / INRF2 is broadly expressed, with highest levels in skeletal muscle. KEAP1 / INRF2 is a key regulator of the NRF2 transcription factor, which transactivates the antioxidant response element (ARE) and upregulates numerous proteins involved in antioxidant defense. Under basal conditions, KEAP1 / INRF2 targets NRF2 for ubiquitination and proteolytic degradation and as such is responsible for the rapid turnover of NRF2. KEAP1 / INRF2 retains NFE2L2 / NRF2 in the cytosol. KEAP1 / INRF2 functions as substrate adapter protein for the E3 ubiquitin ligase complex formed by CUL3 and RBX1. It targets NFE2L2 / NRF2 for ubiquitination and degradation by the proteasome, thus resulting in the suppression of its transcriptional activity and the repression of antioxidant response element-mediated detoxifying enzyme gene expression. KEAP1 / INRF2 may also retain BPTF in the cytosol. It targets PGAM5 for ubiquitination and degradation by the proteasome.

Human KEAP1 References
  • Strachan GD. et al., 2004, Biochemistry. 43: 12113-22.
  • Zhang DD. et al., 2004, Mol Cell Biol. 24: 10941-53.
  • Zhang DD. et al., 2003, Mol Cell Biol. 23: 8137-51.
  • Lo S-C. et al., 2006, J Biol Chem. 281: 37893-903.
  • Buckley BJ. et al., 2008, Free Radic Biol Med. 44 (4) :692-8.
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    Catalog: 11981-H29BL-300
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