Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling FOXP4 with ab242127 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling FOXP4 with ab242127 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human tonsil. The section was incubated with ab242127 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 cells labelling FOXP4 with ab242127 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HepG2 cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling FOXP4 with ab242127 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse spleen. The section was incubated with ab242127 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling FOXP4 with ab242127 at 1/4000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat spleen. The section was incubated with ab242127 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• WB

Lab

Western blot - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

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

Bands around 50 kDa and 37 kDa in lane3 are caused by degradation.

Exposure time : 3 minutes

All lanes:

Western blot - Anti-FOXP4 antibody [EPR22714-2] (<a href='/en-us/products/primary-antibodies/foxp4-antibody-epr22714-2-ab242127'>ab242127</a>) at 1/1000 dilution

Lane 1:

Human colon tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Lane 3:

Rat spleen tissue lysate at 20 µg

Secondary

Lanes 1 - 2:

VeriBlot for IP secondary antibody(HRP)(<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lane 3:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 73 kDa

Observed band size: 80 kDa

false

• WB

Lab

Western blot - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

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

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time : 3 minutes

All lanes:

Western blot - Anti-FOXP4 antibody [EPR22714-2] (<a href='/en-us/products/primary-antibodies/foxp4-antibody-epr22714-2-ab242127'>ab242127</a>) at 1/1000 dilution

Lane 1:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma ) 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 1/100000 dilution

Predicted band size: 73 kDa

Observed band size: 80 kDa

false

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse colon tissue labeling FOXP4 with ab242127 at 1/1000 dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Nuclear staining on mouse colon is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IP

Supplier Data

Immunoprecipitation - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

FOXP4 was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab242127 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab242127 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate 10 ug

Lane 2 : ab242127 IP in HepG2 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab242127 in HepG2 whole cell lysate

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

Exposure time : 8 seconds

All lanes:

Immunoprecipitation - Anti-FOXP4 antibody [EPR22714-2] (<a href='/en-us/products/primary-antibodies/foxp4-antibody-epr22714-2-ab242127'>ab242127</a>)

Predicted band size: 73 kDa

Observed band size: 80 kDa

false

• WB

Lab

Western blot - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

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

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time : Lanes 1-2 : 59 seconds; Lanes 3-4 : 3 minutes.

All lanes:

Western blot - Anti-FOXP4 antibody [EPR22714-2] (<a href='/en-us/products/primary-antibodies/foxp4-antibody-epr22714-2-ab242127'>ab242127</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Hepa1-6 (mouse hepatoma 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 1/100000 dilution

Predicted band size: 73 kDa

Observed band size: 80 kDa

false

• WB

Lab

Western blot - Anti-FOXP4 antibody [EPR22714-2] - BSA and Azide free (AB279342)

This data was developed using ab242127, the same antibody clone in a different buffer formulation.

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

The lysates were kindly provided by our collaborator Dr. Bin Zhao, Zhejiang University.

Lanes 1-2 : Merged signal (red and green). Green - ab242127 observed at 80 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

Lanes 1-2 : ab242127 Anti-FOXP4 antibody [EPR22714-2] was shown to react with FOXP4 in HepG2 cells in Western blot. Loss of signal was observed when FOXP4 knockout sample was used. Wild-type and FOXP4 knockout samples were subjected to SDS-PAGE. ab242127 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 dilution and 1 in 10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-FOXP4 antibody [EPR22714-2] (<a href='/en-us/products/primary-antibodies/foxp4-antibody-epr22714-2-ab242127'>ab242127</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

FOXP4 knock out HepG2 whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 73 kDa

Observed band size: 80 kDa

false