Cat. # | Size | Qty. | Price |
---|---|---|---|
8047S | 100 µl |
|
REACTIVITY | H M R |
SENSITIVITY | Endogenous |
MW (kDa) | 60-64 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunofluorescence (Immunocytochemistry) | 1:100 - 1:400 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
Human, Mouse, Rat
Monkey, Bovine, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu593 of human KEAP1 protein.
The nuclear factor-like 2 (NRF2) transcriptional activator binds antioxidant response elements (ARE) of target gene promoter regions to regulate expression of oxidative stress response genes. Under basal conditions, the NRF2 inhibitor INrf2 (also called KEAP1) binds and retains NRF2 in the cytoplasm where it can be targeted for ubiquitin-mediated degradation (1). Small amounts of constitutive nuclear NRF2 maintain cellular homeostasis through regulation of basal expression of antioxidant response genes. Following oxidative or electrophilic stress, KEAP1 releases NRF2, thereby allowing the activator to translocate to the nucleus and bind to ARE-containing genes (2). The coordinated action of NRF2 and other transcription factors mediates the response to oxidative stress (3). Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD) (4). NRF2 activity in lung cancer cell lines directly correlates with cell proliferation rates, and inhibition of NRF2 expression by siRNA enhances anti-cancer drug-induced apoptosis (5).
KEAP1 contains an amino terminal BTB/POZ domain and a carboxyl terminal KELCH domain (6,7). The KELCH domain is required for interaction with NRF2, and the BTB/POZ domain functions in binding Cul3 E3 ubiquitin ligase (8-10). Under normal conditions, the complex leads to the cytoplasmic sequestration and ubiquitin-mediated proteasomal degradation of NRF2. Electrophilic modification of KEAP1 leads to disassociation of the NRF2/KEAP1 complex. KEAP1 also targets the down regulation of NF-κB activity by targeting IKKβ degradation (11). Mutation of the corresponding KEAP1 gene is seen in lung cancer cases and can lead to uncontrolled activation of NRF2 (12-14).
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