Anti-KLF5 antibody [EPR29066-77]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Immunohistochemical analysis of paraffin-embedded A Wild-type A549 (human lung carcinoma epithelial cell) cell pellet B KLF5 knockout A549 cell pellet tissue labeling KLF5 with ab324105 at 1/2000 (0.244 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on A : Wild-type A549 cell pellet, no staining on B : KLF5 knockout A549 cell pellet. The section was incubated with ab324105 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized KLF5 KO A549 (KLF5 knockout human lung carcinoma epithelial cell), ab301031 cells labelling KLF5 with ab324105 at 1/100 (4.87 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing nuclear staining in wildtype A549 cells (shown in green), showing no staining in KLF5 knockout A549 cells. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/ab195889 200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ab150081 1000 2ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling KLF5 with ab324105 at 1/500 (0.974 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The section was incubated with ab324105 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling KLF5 with ab324105 at 1/500 (0.974 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon carcinoma. The section was incubated with ab324105 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-KLF5 antibody [EPR29066-77] (AB324105)

KLF5 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab324105 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324105 at 1/1000 dilution.

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-KLF5 antibody [EPR29066-77] (ab324105) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab324105 at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324105 in HeLa whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 50 kDa

false

Exposure time: 58s

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab324105 [EPR29066-77]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab324105 [EPR29066-77]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 µg of ab324105 [EPR29066-77]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling KLF5 with ab324105 at 1/2000 (0.244 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon. The section was incubated with ab324105 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized ES-D3 (mouse blastocyst-derived embryonic stem cell) cells labelling KLF5 with ab324105 at 1/100 (4.87 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ab150081 1000 2ug/ml dilution (Green).

Confocal image showing nuclear staining in ES-D3 [D3] cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/ab195889 200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ab150081 1000 2ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Immunohistochemical analysis of paraffin-embedded Mouse pancreatic carcinoma tissue labeling KLF5 with ab324105 at 1/2000 (0.244 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse pancreatic carcinoma. The section was incubated with ab324105 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).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Supplier Data

Western blot - Anti-KLF5 antibody [EPR29066-77] (AB324105)

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

The bands beneath the target band (50 kDa) are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-KLF5 antibody [EPR29066-77] (ab324105) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeting KLF5 whole cell lysate at 20 µg

Lane 3:

SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa,36 kDa

false

Exposure time: 59s

• Dot

Supplier Data

Dot Blot - Anti-KLF5 antibody [EPR29066-77] (AB324105)

Dot blot analysis of KLF5 using ab324105 at 1 : 1000 (0.487 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 10,000 dilution.

Lane1 : His-tagged human KLF5 recombinant protein
Lane2 : His-tagged human KLF2 recombinant protein
Lane3 : His-tagged human KLF3 recombinant protein
Lane4 : His-tagged human KLF4 recombinant protein
Lane5 : LIF-tagged human KLF1 recombinant protein

Exposure time : 70 seconds.

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

This antibody does not cross-react with human KLF1, KLF2, KLF3 and KLF4.

In Dot Blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.#In Dot Blot, Anti-LIF antibody [EPR21713-25] - BSA and Azide free staining at Capture dilution.

All lanes:

Dot Blot - Anti-KLF5 antibody [EPR29066-77] (ab324105) at 1/1000 dilution

Lane 1:

His-tagged human KLF5 recombinant protein

Lane 2:

His-tagged human KLF2 recombinant protein

Lane 3:

His-tagged human KLF3 recombinant protein

Lane 4:

His-tagged human KLF4 recombinant protein

Lane 5:

LIF-tagged human KLF1 recombinant protein

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

false

Exposure time: 70s

• WB

Supplier Data

Western blot - Anti-KLF5 antibody [EPR29066-77] (AB324105)

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

In lane1, to minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The bands beneath the target band (50 kDa) are likely to be degraded target fragments.

All lanes:

Western blot - Anti-KLF5 antibody [EPR29066-77] (ab324105) at 1/1000 dilution

Lane 1:

ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) whole cell lysate (Fresh lysates) at 20 µg

Lane 2:

ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) whole cell lysate (Frozen lysates) at 20 µg

Lane 3:

Mouse E7 embryo tissue lysate (Frozen lysates) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa

false

Exposure time: 158s