Anti-ATF6 antibody [EPR22690-84] - ChIP Grade

6 product images

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  ChIP - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

  Chromatin was prepared from HeLa cells treated with thapsigargin (1 uM, 1 h) according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
  The ChIP was performed with 25 μg of chromatin, 5 μg of ab227830 (red), or 5 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 20 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
  Primers and probes are from paper PMID: 17535801.

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  Immunoprecipitation - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

  ATF6 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab227830 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab227830 1/1000 dilution (0.5 μg/ml). VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  

  Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg

  Lane 2: Anti-IGFBP5 antibody [EPR18013-137] ab254324 IP in HeLa whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab227830 in HeLa whole cell lysate.

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

  Exposure time: 3 min.

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

  All lanes: Immunoprecipitation - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

  Predicted band size: 74 kDa

  Observed band size: 95 kDa

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  Western blot - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

  ab227830 was shown to specifically react with ATF6 in wild-type HAP1 cells as signal was lost in ATF6 knockout cells. Wild-type and ATF6 knockout samples were subjected to SDS-PAGE. ab227830 and Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) secondary antibody at 1/20,000 dilution for 1 hour at room temperature before imaging.
  
  Exposure time 62 seconds.
  
  Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

  All lanes: Western blot - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830) at 1/1000 dilution

  Lane 1: Wild type HAP1 whole cell lysate at 20 µg

  Lane 2: ATF6 knockout HAP1 whole cell lysate at 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

  Predicted band size: 74 kDa

  Observed band size: 90 kDa

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  Western blot - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

  ATF6 is cleaved upon ER stress and the molecular weight observed is consistent with what has been described in the literature (PMID: 25149687; 11163209).
  
  Exposure time 3 minutes.
  
  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  All lanes: Western blot - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830) at 1/1000 dilution

  Lane 1: Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 10 µg

  Lane 2: HeLa treated with 1 μΜ thapsigargin for 1 hour, whole cell lysate at 10 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

  Predicted band size: 74 kDa

  Observed band size: 50 kDa, 90 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

  Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling ATF6 with ab227830 at 1/1000 dilution (0.566 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Positive staining on human kidney (PMID: 25725420) is observed. The section was incubated with ab227830 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 (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

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  ChIC/CUT&RUN sequencing - Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

  ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HeLa cells treated with Thapsigargin (0.5 µM for 24h) and 5µg of ab227830 [EPR22690-84]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.