Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T cells labelling CD137 with ab243648 at 1/100 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) antibody at 1/1000 dilution (Red). Confocal image showing cytoplasmic and membranous staining in HEK-293T cells transfected with a GFP-tagged CD137 expression vector -  the GPF tag is shown in Green. The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution.

All lanes : Anti-CD137 antibody [EPR23218-111] (ab243648) at 1/1000 dilution

Lane 1 : Untreated mouse splenocytes, whole cell lysate
Lane 2 : Mouse splenocytes (treated with 2.5 µg/ml Concanavalin A for 72 hours), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 28 kDa
Observed band size: 21-24,36-40 kDa why is the actual band size different from the predicted?

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

CD137 is a type I transmembrane glycoprotein and has both monomeric and dimeric forms on the surface of activated T cells (PMID: 30355497).

Exposure time: 3 minutes.

Flow cytometric analysis of Mouse splenocytes (treated with 2.5ug/ml concanavalin A  for 72h) cells labelling CD137 with ab243648 at 1/500 (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG (Left) or ab243648 (Right), then stained with anti-CD25 conjugated to BV421.

Gated on viable cells.

All lanes : Anti-CD137 antibody [EPR23218-111] (ab243648) at 1/1000 dilution

Lane 1 : His-tagged mouse CD137 recombinant protein(aa24-187), 10 ng
Lane 2 : His-tagged human CD137 recombinant protein(aa24-186), 10 ng

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

Predicted band size: 28 kDa

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

Exposure time: 15 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Mouse splenocytes cells labelling CD137 with ab243648 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and membranous staining in mouse splenocytes treated with Concanavalin A (2.5 ug/ml) for 72 h. ab195889 Anti-alpha Tubulin antibody [DM1A] - 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.

CD137 was immunoprecipitated from 0.35 mg mouse splenocyte (treated with 2.5 μg/ml Concanavalin A for 72 hours) whole cell lysate with ab243648 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab243648 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse splenocyte (treated with 2.5 μg/ml Concanavalin A for 72 hours) whole cell lysate 10 μg

Lane 2: ab243648 IP in Mouse splenocyte (treated as above) whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab243648 in Mouse splenocyte (treated as above) whole cell lysate

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

Exposure time: 76 seconds.

CD137 is a type I transmembrane glycoprotein and has both monomeric and dimeric forms on the surface of activated T cells (PMID: 30355497).