Anti-Egr1 抗体 [EPR23981-46] (ab300449)

Egr1 was immunoprecipitated from 0.35 mg PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate with ab300449 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab300449. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution

Lane 1: PC-12 (rat adrenal gland pheochromocytoma cell) starved overnight, then treated with 100ng/ml NGF and 10uM MG-132 for 2 hours, whole cell lysate 10 μg (Input).
Lane 2: PC-12 starved overnight, then treated with 100ng/ml NGF and 10uM MG-132 for 2 hours, whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300449 in PC-12 starved overnight, then treated with 100ng/ml NGF and 10uM MG-132 for 2 hours, whole cell lysate (-).

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

Lysate was freshly made and used immediately to minimize protein degradation.

The bands beneath the target band (80 kDa) are likely to be degraded target fragments.

Egr1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab300449 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab300449. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution

Lane 1: NIH/3T3 (mouse embryonic fibroblast) starved overnight, then treated with 10ng/ml EGF and 10uM MG-132 for 2 hours, whole cell lysate 10 μg (Input).
Lane 2: NIH/3T3 starved overnight, then treated with 10ng/ml EGF and 10uM MG-132 for 2 hours, whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300449 in NIH/3T3 starved overnight, then treated with 10ng/ml EGF and 10uM MG-132 for 2 hours, whole cell lysate (-).

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

Lysate was freshly made and used immediately to minimize protein degradation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) cells labelling Egr1 with ab300449 at 1/100 dilution (5.48 μg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly nuclear staining in PC-12 cells, which were first starved for 18 hours, then treated with EGF (10 ng/ml) and MG-132(10 uM) for 2 h. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used as counterstain at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Egr1 with ab300449 at 1/100 dilution (5.48 μg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly nuclear staining in NIH/3T3 cells, which were first starved for 18 hours, then treated with EGF (10 ng/ml) and MG-132(10 uM) for 2 h. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used as counterstain at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

All lanes : Anti-Egr1 antibody [EPR23981-46] (ab300449) at 1/1000 dilution

Lane 1 : Untreated PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate
Lane 2 : PC-12 starved overnight, then treated with 100ng/ml NGF and 10uM MG-132 for 2 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 57 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

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

ab181602 was used as loading control for GAPDH.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes : Anti-Egr1 antibody [EPR23981-46] (ab300449) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate
Lane 2 : THP-1 treated with 100ng/ml LPS (lipopolysaccharide) for 3 hours whole cell lysate
Lane 3 : Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 4 : NIH/3T3 starved overnight, then treated with 10ng/ml EGF and 10uM MG-132 for 2 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 57 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

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

ab181602 was used for GAPDH loading control.

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

The identity of the lower MW band at approximately 20 kDa is unknown.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labelling Egr1 with ab300449 at 1/500 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection kit). Positive staining on human cerebrum (PMID: 11106242). The section was incubated with ab300449 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by a ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse hippocampus tissue labelling Egr1 with ab300449 at 1/2000 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection kit). Positive staining on mouse hippocampus. The section was incubated with ab300449 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by a ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue labelling Egr1 with ab300449 at 1/2000 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection kit). Positive staining on rat hippocampus. The section was incubated with ab300449 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by a ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labelling Egr1 with ab300449 at 1/2000 followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection kit).

Negative control: No staining on mouse kidney (PMID: 10961876).

The section was incubated with ab300449 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody followed by a ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cortex (fresh) tissue labeling Egr1 with ab300449 at 1/100 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (Green). Positive staining on mouse cortex is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution. 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver (fresh) tissue labeling Egr1 with ab300449 at 1/100 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (Green).

Negative control (PMID: 10961876). No staining on mouse liver is observed.

The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.