Anti-KEAP1 Polyclonal Antibody 2 is a Rabbit antibody targeting KEAP1. Anti-KEAP1 Polyclonal Antibody 2 can be used in FCM, ICC/IF, IF, IHC-Fr, IHC-P, WB.

IgG

Human,Mouse,Rat (predicted:Dog,Pig,Cow,Rabbit)

1. Sample:
A673 (Human) Cell Lysate at 30 μg
A431 (Human) Cell Lysate at 30 μg
Primary: Anti-KEAP1 (TMAB-01018) at 1/500 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 69 kDa
Observed band size: 69 kDa
2. Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 min and DAB staining.
3. Paraformaldehyde-fixed, paraffin embedded (Human cervical carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 min and DAB staining.
4. Paraformaldehyde-fixed, paraffin embedded (Human kidney); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:400 overnight at 4°C, followed by a conjugated secondary antibody for 20 min and DAB staining.
5. Paraformaldehyde-fixed, paraffin embedded (rat skin); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
6. Paraformaldehyde-fixed, paraffin embedded (mouse kidney); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
7. Paraformaldehyde-fixed, paraffin embedded (rat kidney); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
8. Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
9. Paraformaldehyde-fixed, paraffin embedded (mouse skin); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
10. Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (KEAP1) Polyclonal Antibody, Unconjugated (TMAB-01018) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
11. A-431 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (KEAP1) polyclonal Antibody, Unconjugated (TMAB-01018) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
12. A-549cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (KEAP1) polyclonal Antibody, Unconjugated (TMAB-01018) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
13. Blank control (black line): A549. Primary Antibody (green line): Rabbit Anti-KEAP1 antibody (TMAB-01018)
Dilution: 1 μg/Test;
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF488
Dilution: 0.5 μg/Test.
Isotype control (orange line): Normal Rabbit IgG
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C, The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature.

FCM=1 μg/Test; ICC/IF=1:100-500; IF=1:100-500; IHC-Fr=1:100-500; IHC-P=1:100-500; WB=1:500-2000

Polyclonal

KLH conjugated synthetic peptide: human KEAP1

Human

KEAP1

Kelch-like ECH-associated protein 1

Ubiquitylation

Retains NFE2L2/NRF2 in the cytosol. Functions as substrate adapter protein for the E3 ubiquitin ligase complex formed by CUL3 and RBX1. 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. May also retain BPTF in the cytosol. Targets PGAM5 for ubiquitination and degradation by the proteasome.