Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529] (AB326338)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Parental HAP1 (human chronic myelogenous leukemia near-haploid cell) and KEAP1 KO HAP1 (KEAP1 knockout human chronic myelogenous leukemia near-haploid cell) cells labelling with ab326338 at 1/50 (9.82 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in parental HAP1 cell line and no staining in KEAP1 KO HAP1 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529] (AB326338)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellet and (B) KEAP1 knockout HAP1 cell pellet tissue labeling with ab326338 at 1/200 (2.455 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) Wild-type HAP1 cell pellet, no staining on (B) KEAP1 knockout HAP1 cell pellet.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529] (AB326338)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling with ab326338 at 1/100 (4.91 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human kidney.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529] (AB326338)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) (Green and black) and KEAP1 KO HAP1(HO010289) (Magenta and grey) cells labelling with ab326338 at 1/500 dilution (0.1ug), compared with a Rabbit monoclonal IgG (ab172730) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529] (AB326338)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, ab326338 was shown to bind specifically to KEAP1.
Target of interest was observed at 70 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in HAP1 knockout cell line (lane 2).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Kelch-like ECH-associated protein 1 antibody [EPR31136-529] (ab326338) at 1/1000 dilution

Lane 1:

Wild-type HAP-1 (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate at 60 µg

Lane 2:

KEAP1 knockout HAP1 whole cell lysate at 60 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 60 µg

Lane 4:

Calu-1 (human lung epidermoid carcinoma epithelial cell) whole cell lysate at 60 µg

Lane 5:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 60 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 70 kDa,36 kDa

false

Exposure time: 37s