Anti-c-Jun antibody [E254] - ChIP Grade

7 product images

• 

  ChIP - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  Chromatin was prepared from PC-12 (starve overnight) + NGF ( 50 ng/ml 2h) cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab32137 (blue), and 20µl of protein A/G sepharose beads slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 5µg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• 

  Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  ab32137 (purified) at 1/500 dilution (2.29 ©:g/ml) immunoprecipitating c-Jun in HEK-293 whole cell lysate.
  Lane 2 (+): HEK-293(Human embronic kidney epithelial cell) whole cell lysate 10 ©:g
  Lane 3 (-): ab32137 & HEK-293 whole cell lysateRabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32137 in HEK-293 whole cell lysate
  For western blotting, VeriBlot for IP secondary antibody (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used as the secondary antibody at 1/1000 dilution.
  Blocking and diluting buffer: 5% NFDM /TBST .

  All lanes: Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  Predicted band size: 35 kDa

• 

  Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  c-Jun was immunoprecipitated using 0.5mg NIH3T3 whole cell extract, 5µg of Rabbit polyclonal to c-Jun and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
  The antibody was incubated under agitation with Protein G beads for 10min, NIH3T3 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
  Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab32137.
  Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) ab99697).
  Band: 45kDa; c-Jun

  All lanes: Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  Predicted band size: 35 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling c-Jun with purified ab32137 at 1:100 dilution (1.2 µg/ml). Heat mediated antigen retrieval was performed using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• 

  Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  Blocking and diluting buffer: 5% NFDM/TBST
  

  For Lysate preparation protocol, please refer to the protocol section of the website and/or here.

  All lanes: Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137) at 1/200 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method at 15 µg

  Lane 2: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates prepared in 1% SDS Hot lysis method. at 15 µg

  Lane 3: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates prepared in RIPA lysis method at 15 µg

  Lane 4: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates prepared in 1% SDS Hot lysis method 15ug. at 15 µg

  Secondary

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

  Predicted band size: 35 kDa

  Observed band size: 39 kDa

  Exposure time: 3min

• 

  Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  All lanes: Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137) at 1/5000 dilution

  All lanes: PC-12 (Rat adrenal gland pheochromocytoma) 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 1/20000 dilution

  Predicted band size: 35 kDa

  Observed band size: 43 kDa

• 

  OI-RD Scanning - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

  We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
  Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.