Anti-Egr1 antibody [EPR23981-203] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (AB307199)

Immunohistochemical analysis of paraffin-embedded Rat hippocampus tissue lABeling Egr1 with ab307199 at 1/2000 (0.275 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 ab307199 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (AB307199)

Immunohistochemical analysis of paraffin-embedded Rat liver tissue lABeling Egr1 with ab307199 at 1/2000 (0.275 ug/ml) followed by a ready to use LeicaDS9800 (Bond? Polymer Refine Detection) was used. Negative control : no staining on rat liver.The section was incubated with ab307199 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (AB307199)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeABilized PC-12 (rat adrenal gland pheochromocytoma cell) cells lABelling Egr1 with ab307199 at 1/50 (11 ug/ml) dilution, followed by ab150081 Goat Anti-RABbit IgG H&L (Alexa Fluor? 488) preadsorbed antibody at 1/1000 2 ug/ml 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). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor? 594) was used to counterstain tubulin at 1/200 2.5ug/ml 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 2 ug/ml dilution.

• ChIP

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ChIP - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (AB307199)

Chromatin was prepared from PC-12 (starve overnight) treated with NGF (50ng/ml 1h) and PC-12 (starve overnight) non-treated according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min. The ChIP was performed with 25 µg of chromatin, 5 µg of ab307199 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers are from paper PMID : 28076410 *http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• IP

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Immunoprecipitation - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (AB307199)

Egr1 was immunoprecipitated from 0.35 mg 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 5 ug with ab307199 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307199 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 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 5 ug Lane 2 : ab307199 IP in 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 ab307199 in PC-12 starved overnight, then treated with 100ng/ml NGF and 10uM MG-132 for 2 hours, whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 15 seconds Lysate was freshly made and used immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (ab307199) at 1/1000 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 5 μg

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 ab307199 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

Observed band size: 80 kDa

false

Exposure time: 15s

• WB

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Western blot - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (AB307199)

Blocking and diluting buffer and concentration : 5% NFDM/TBSTLysates were freshly made and used for Western blotting immediately to minimize protein degradation. Exposure time : Lane 1 : 114 seconds; Lane 2 : 92 seconds; Lane 3 : 15 seconds.

All lanes:

Western blot - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (ab307199) at 1/1000 dilution

Lane 1:

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

Lane 2:

PC-12 starved overnight, then treated with /ml NGF and 10uM MG-132 for 2 hours whole cell lysate 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 1/100000 dilution

Observed band size: 80 kDa

false

Exposure time: 15s