Lane 1 : Anti-FOXP3 antibody [EPR22102-37] (ab215206) at 1/200 dilution
Lane 2 : Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution (Anti-DDDDK tag, 1:1000 dilution)

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

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

Blocking buffer and concentration: 5% NFDM /TBST

Flow cytometric analysis of 4% 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.

Immunohistochemical analysis of paraffin-embedded human thymus tissue labeling FOXP3 with ab215206 at 1/1000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on regulatory T-cells in human thymus (PMID: 16380964) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab208572 (Universal HIER antigen retrieval reagent).

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 : 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
Lane 2 : HEK-293T transfected with FOXP3 (WT) expression vector containing a GFP-myc tag, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?


Exposure time: 6 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

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).

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.

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.

Immunohistochemical analysis of paraffin-embedded rat thymus tissue labeling FOXP3 with ab215206 at 1/1000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on regulatory T-cells in rat thymus (PMID: 16380964) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab208572 (Universal HIER antigen retrieval reagent).

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling FOXP3 with ab215206 at 1/1000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on regulatory T-cells in mouse spleen (PMID: 16380964) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab208572 (Universal HIER antigen retrieval reagent).

Immunohistochemical analysis of paraffin-embedded mouse thymus tissue labeling FOXP3 with ab215206 at 1/1000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on regulatory T-cells in mouse thymus (PMID: 16380964) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab208572 (Universal HIER antigen retrieval reagent).

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling FOXP3 with ab215206 at 1/1000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on regulatory T-cells in human spleen (PMID: 16380964) is observed. Counter stained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval using ab208572 (Universal HIER antigen retrieval reagent).