Anti-FOXP3 antibody [EPR22102-37]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Intracellular flow cytometric analysis of4% paraformaldehyde-fixed, 90% methanol-permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell line transfected with a GFP-tagged FOXP3 expression construct labeling FOXP3 with ab215206 at 1/40 dilution (Right) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 647) (ab150079), at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Human PBMCs were stained with Alexa Fluor^® 647 conjugated anti-human CD4 and BV421 conjugated anti-human CD25. Cells were then fixed with 2% PFA for 10min and permeabilized with True-Nuclear™ permeabilization buffer, followed by intracellular staining with rabbit IgG (ab172730, Left) or anti-FOXP3 RabMab (Right, 1/40). ab98462, Dylight^®488-conjugated goat anti-rabbit IgG was used as the secondary at a dilution of 1/2000.

Gating strategy and expression pattern is consistent with literature (PMID : 27330808).

• IP

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Immunoprecipitation - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

FOXP3 was immunoprecipitated from 0.35 mg of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate with ab215206 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab215206 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : HEK-293T cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate 10 μg (Input).
Lane 2 : ab215206 IP in HEK-293T cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate (+).
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab215206 in HEK-293T cells transfected with a GFP-tagged FOXP3 expression construct whole cell lysate (-).

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 seconds.

FOXP3 isoforms have been described in the literature. (PMID : 19568423).

All lanes:

Immunoprecipitation - Anti-FOXP3 antibody [EPR22102-37] (ab215206)

Predicted band size: 47 kDa,90 kDa

Observed band size: 55-73 kDa

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• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Rat splenocytes were stained with Alexa Fluor^® 647 conjugated anti-rat CD4. Cells were then fixed with 2% PFA for 10min and permeabilized with True-Nuclear™ permeabilization buffer, followed by intracellular staining with rabbit IgG (ab172730, Left) or anti-FOXP3 RabMab (Right, 1/40). ab150077, Alexa Fluor^®488-conjugated goat anti-rabbit IgG was used as the secondary at a dilution of 1/2000.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Mouse splenocytes were stained with Alexa Fluor^® 647 conjugated anti-mouse CD4. Cells were then fixed with 2% PFA for 10min and permeabilized with True-Nuclear™ permeabilisation buffer, followed by intracellular staining with rabbit IgG (ab172730, Left) or anti-FOXP3 RabMab (Right, 1/40). ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG was used as the secondary at a dilution of 1/2000.

• WB

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Western blot - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FOXP3 antibody [EPR22102-37] (ab215206) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a GFP-myc tag, whole cell lysate at 10 µg

Lane 2:

HEK-293T transfected with FOXP3 (WT) expression vector containing a GFP-myc tag, whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 73 kDa

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Exposure time: 6s

• WB

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Western blot - Anti-FOXP3 antibody [EPR22102-37] (AB215206)

Blocking buffer and concentration : 5% NFDM /TBST

Lane 1:

Western blot - Anti-FOXP3 antibody [EPR22102-37] (ab215206) at 1/200 dilution

Lane 2:

Western blot - Anti-FOXP3 antibody [EPR22102-37] (ab215206) at 1/1000 dilution

All lanes:

Full-length C-MYC/DDK tagged Recombinant Mouse Foxp3 protein 10ng

Secondary

All lanes:

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

Predicted band size: 47 kDa

Observed band size: 50 kDa

false

Exposure time: 180s