All lanes : Anti-ATF2 antibody [EPR22938-114] (ab239361) at 1/1000 dilution

Lane 1 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 2 : Rat spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 70 kDa why is the actual band size different from the predicted?

The molecular weight and degraded fragments observed are consistent with what has been described in the literature (PMID: 9488727, 10207054, 26901653).  Suggest use freshly made lysate to minimize protein degradation.

Exposure time: 3 minutes.

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST.

Anti-ATF2 antibody [EPR22938-114] (ab239361) at 1/1000 dilution + Mouse brain tissue lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 70 kDa why is the actual band size different from the predicted?

The molecular weight observed is consistent with what has been described in the literature (PMID:26901653).

Exposure time: 3 minutes.

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-ATF2 antibody [EPR22938-114] (ab239361) at 1/1000 dilution

Lane 1 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 3 : HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 4 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 5 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 70 kDa why is the actual band size different from the predicted?

The molecular weight observed is consistent with what has been described in the literature (PMID:26901653).

Exposure time: 10 seconds.

Blocking and Diluting Buffer and concentration: 5% NFDM/TBST.

ATF2 was immunoprecipitated from 0.35 mg NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate with ab239361 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab239361. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10µg.

Lane 2: ab239361 IP in NIH/3T3 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab239361 in NIH/3T3 whole cell lysate.

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

Exposure time: 8 seconds

Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.

The molecular weight and degraded fragments observed are consistent with what has been described in the literature (PMID: 9488727, PMID: 10207054, PMID:26901653).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labeling ATF2 with ab239361 at 1/40 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling ATF2 with ab239361 at 1/40 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling ATF2 with ab239361 at 1/250 dilution (1.76 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat kidney is observed. The section was incubated with ab239361 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: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling ATF2 with ab239361 at 1/250 dilution (1.76 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse kidney is observed. The section was incubated with ab239361 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: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded human kidney carcinoma tissue labeling ATF2 with ab239361 at 1/250 dilution (1.76 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human kidney carcinoma (PMID: 27377902) is observed. The section was incubated with ab239361 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: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling ATF2 with ab239361 at 1/250 dilution (1.76 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human kidney (PMID: 27377902) is observed. The section was incubated with ab239361 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).