Anti-Egr1 antibody [EPR23981-203] - ChIP Grade - BSA and Azide free

6 product images

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  ChIP - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade - BSA and Azide free (ab307200)

  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 Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199 (red), or 5 µg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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

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  Western blot - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade - BSA and Azide free (ab307200)

  This data was developed using 307199, the same antibody clone in a different buffer formulation.
  Blocking and diluting buffer and concentration: Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.
  Lane 1: 114 seconds;
  Lane 2: 92 seconds;
  Lane 3: 15 seconds.
  Exposure time:

  All lanes: Western blot - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade (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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 80 kDa

  Exposure time: 15s

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  Immunoprecipitation - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade - BSA and Azide free (ab307200)

  This data was developed using Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199, the same antibody clone in a different buffer formulation.
  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 Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (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: ABAB307199 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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-Egr1 antibody [EPR23981-203] - ChIP Grade 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 (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: Immunoprecipitation - Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730)

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 80 kDa

  Exposure time: 15s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade - BSA and Azide free (ab307200)

  This data was developed using Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199, the same antibody clone in a different buffer formulation.
  Immunohistochemical analysis of paraffin-embedded Rat hippocampus tissue labeling Egr1 with Anti-Egr1 antibody [EPR23981-203] - ChIP Grade 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 Anti-Egr1 antibody [EPR23981-203] - ChIP Grade 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

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade - BSA and Azide free (ab307200)

  This data was developed using Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199, the same antibody clone in a different buffer formulation.
  Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Egr1 with Anti-Egr1 antibody [EPR23981-203] - ChIP Grade 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 Anti-Egr1 antibody [EPR23981-203] - ChIP Grade 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

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  Immunocytochemistry/ Immunofluorescence - Anti-Egr1 antibody [EPR23981-203] - ChIP Grade - BSA and Azide free (ab307200)

  This data was developed using Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199, 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 Anti-Egr1 antibody [EPR23981-203] - ChIP Grade ab307199 at 1/50 (11 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-RABbit IgG H&L (Alexa Fluor? 488) preadsorbed at 1/1000 2 ug/ml dilution.