Recombinant Anti-Keap1 antibody [EPR22664-26] (ab227828)

All lanes : Anti-Keap1 antibody [EPR22664-26] (ab227828) at 1/1000 dilution

Lane 1 : Human lung tissue lysate
Lane 2 : Wild-type HAP1 whole cell lysate
Lane 3 : Keap1 knockout HAP1 whole cell lysate
Lane 4 : NK-92 (human malignant non-Hodgkin's lymphoma natural killer cell), whole cell lysate
Lane 5 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate
Lane 6 : Mouse E14.5 liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 2-6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa


Exposure time: 3 minutes

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

ab227828 was shown to specifically react with Keap1 in wild-type HAP1 cells as signal was lost in Keap1 knockout cells. Wild-type and Keap1 knockout samples were subjected to SDS-PAGE. ab227828 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 23242280, PMID: 20404090).

This blot was developed using a higher sensitivity ECL substrate.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Keap1 with ab227828 at 1/500 dilution (0.97 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on kidney tubules of mouse kidney (PMID: 28324005) is observed. The section was incubated with ab227828 for 30 mins at room temperature.  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labeling Keap1 with ab227828 at 1/500 dilution (0.97 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on tumor cells of human lung carcinoma (PMID: 20534738) is observed. The section was incubated with ab227828 for 30 mins at room temperature.  The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).