Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Egr1 with ab300449 at 1/500 (1.096 ug/ml) followed by a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used. 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 : Secondary antibody is a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Rat hippocampus tissue labeling Egr1 with ab300449 at 1/2000 (0.274 ug/ml) followed by a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used. 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 : Secondary antibody is a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Egr1 with ab300449 at 1/2000 (0.274 ug/ml) followed by a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used. 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 : Secondary antibody is a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (fresh) tissue labeling Egr1 with ab300449 at 1/100 (5.48 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL 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 2 ug/mL dilution. .

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cortex (fresh) tissue labeling Egr1 with ab300449 at 1/100 (5.48 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL 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 2 ug/mL dilution. .

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation. 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation. 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Mouse hippocampus tissue labeling Egr1 with ab300449 at 1/2000 (0.274 ug/ml) followed by a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used. 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 : Secondary antibody is a ready to use LeicaDS9800 (BOND® Polymer Refine Detection) was used.Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation. 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.

All lanes:

Immunoprecipitation - Anti-Egr1 antibody [EPR23981-46] (<a href='/en-us/products/primary-antibodies/egr1-antibody-epr23981-46-ab300449'>ab300449</a>) at 1/30 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 at 10 µg

Lane 2:

NIH/3T3 starved overnight, then treated with 10ng/ml EGF and 10uM MG-132 for 2 hours, whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 57 kDa

Observed band size: 80 kDa

false

Exposure time: 24s

• IP

Supplier Data

Immunoprecipitation - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation. 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.

All lanes:

Immunoprecipitation - Anti-Egr1 antibody [EPR23981-46] (<a href='/en-us/products/primary-antibodies/egr1-antibody-epr23981-46-ab300449'>ab300449</a>) at 1/30 dilution

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 (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/egr1-antibody-epr23981-46-ab300449'>ab300449</a> in PC-12 starved overnight, then treated with 100ng/ml NGF and 10uM MG-132 for 2 hours, whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 57 kDa

Observed band size: 80 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation. 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.

All lanes:

Western blot - Anti-Egr1 antibody [EPR23981-46] (<a href='/en-us/products/primary-antibodies/egr1-antibody-epr23981-46-ab300449'>ab300449</a>) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

THP-1 treated with 100ng/ml LPS (lipopolysaccharide) for 3 hours whole cell lysate at 20 µg

Lane 3:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

NIH/3T3 starved overnight, then treated with 10ng/ml EGF and 10uM MG-132 for 2 hours whole cell lysate at 20 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

Predicted band size: 57 kDa

Observed band size: 80 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Egr1 antibody [EPR23981-46] (BSA and Azide free) (AB300450)

This data was developed using ab300449, the same antibody clone in a different buffer formulation. 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:

Western blot - Anti-Egr1 antibody [EPR23981-46] (<a href='/en-us/products/primary-antibodies/egr1-antibody-epr23981-46-ab300449'>ab300449</a>) at 1/1000 dilution

Lane 1:

Untreated PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 2:

PC-12 starved overnight, then treated with 100ng/ml NGF and 10uM MG-132 for 2 hours whole cell lysate at 20 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

Predicted band size: 57 kDa

Observed band size: 80 kDa

false

Exposure time: 15s