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 4ºC. 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

Lanes 1-4 : Anti-KLF4 antibody [EPR20753-25] (ab214666) at 1/1000 dilution
Lane 5 : Anti-KLF4 antibody [EPR20753-25] (ab214666) at 1/5000 dilution

Lane 1 : F9 (mouse embryonal carcinoma epithelial cell), whole cell lysate
Lane 2 : Mouse skin tissue lysate
Lane 3 : Mouse colon tissue lysate
Lane 4 : Rat skin tissue lysate
Lane 5 : ES D3 (mouse embryonic stem cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Lane 5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 54 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

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

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

 

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) (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 (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.

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.

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.

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 (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, 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 (ab150077) at 1/1000.