Anti-KLF4 antibody [EPR20753-25]

6 product images

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  Image courtesy of Vidur Garg from the Sloan-Kettering Institute

  IHC - Wholemount - Anti-KLF4 antibody [EPR20753-25] (ab214666)

  Several embryos at the mid-blastocyst stage (~E3.75) were tested. The embryos were fixed for 10 mins at room temperature in 4% PFA. Fixed embryos were washed for 5 mins in PBS + 0.1% Triton X-100 (PBX) at room temperature. The embryos were then permeabilized in PBS + 0.5% Triton X-100 + 100mM Glycine for 5 mins at room temperature and washed again in PBX for 5 mins at room temperature. Blocking was carried out for 1 hour at room temperature in PBS + 2% horse serum and then the embryos were incubated overnight in ab214666 (1/1000) diluted in blocking solution. The next day embryos were washed three times for 5 mins each in PBX, before blocking again for 1 hour at room temperature. Then incubated in Donkey anti rabbit Alexa Fluor^® 488 secondary antibodies (1/500) diluted in blocking solution for 1 hour at 4°C. This was followed by 2 washes of 5 mins each in PBX. The embryos were then incubated in PBS + 5μg/ml DAPI for 10 min, or until mounting for imaging. Images were acquired on a laser-scanning confocal microscope system. Displayed image shows maximum intensity projection of 5 optical sections acquired at 1μm increments. Scale bar: 20 μm

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  Immunoprecipitation - Anti-KLF4 antibody [EPR20753-25] (ab214666)

  KLF4 was immunoprecipitated from 0.35 mg F9 (Mouse embryonal carcinoma epithelial cell) whole cell lysate using ab214666 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab214666 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used as the secondary at 1/5000 dilution.
  

  Lane 1: F9 whole cell lysate 10 μg (input).
  Lane 2: ab214666 IP in F9 whole cell lysate.
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab214666 in F9 whole cell lysate.

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

  Exposure time: 3 minutes.

  Fresh lysate was required and IP test was conducted immediately. Incubation time was shortened from overnight to 2h.

  All lanes: Immunoprecipitation - Anti-KLF4 antibody [EPR20753-25] (ab214666)

  Predicted band size: 52 kDa, 54 kDa

  Observed band size: 52 kDa, 59 kDa

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  Western blot - Anti-KLF4 antibody [EPR20753-25] (ab214666)

  Exposure times:
  Lanes 1-2: 3 minutes
  Lane 3-4: 92 seconds
  Lane 5: 48 seconds
  

  Blocking/Diluting buffer and concentration: 5% NFDM/TBST

  Lanes 1 - 4: Western blot - Anti-KLF4 antibody [EPR20753-25] (ab214666) at 1/1000 dilution

  Lane 5: Western blot - Anti-KLF4 antibody [EPR20753-25] (ab214666) at 1/5000 dilution

  Lane 1: F9 (mouse embryonal carcinoma epithelial cell), whole cell lysate at 20 µg

  Lane 2: Mouse skin tissue lysate at 20 µg

  Lane 3: Mouse colon tissue lysate at 20 µg

  Lane 4: Rat skin tissue lysate at 20 µg

  Lane 5: ES D3 (mouse embryonic stem cell), whole cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Lane 5: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 54 kDa

  Observed band size: 52 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KLF4 antibody [EPR20753-25] (ab214666)

  Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling KLF4 with ab214666 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Nuclear staining on epithelial cells of rat colon (PMID: 21070761, PMID: 19109406) is observed. Counterstained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

  Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KLF4 antibody [EPR20753-25] (ab214666)

  Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling KLF4 with ab214666 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Nuclear staining on epithelial cells of mouse colon (PMID: 21070761, PMID: 19109406) is observed. Counterstained with Hematoxylin.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

  Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

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  Immunocytochemistry/ Immunofluorescence - Anti-KLF4 antibody [EPR20753-25] (ab214666)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized F9 (mouse embryonal carcinoma epithelial cell) cells labeling KLF4 with ab214666 at 1/100 dilution, followed by a AlexaFluor^®488 Goat anti-Rabbit secondary (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 dilution (Green). Confocal image showing nuclear staining in F9 cell line is observed. The nuclear counterstain was DAPI (Blue). Tubulin was counterstained using an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889, Red) at 1.200 dilution.
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a AlexaFluor^®488 Goat anti-Rabbit secondary (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000.