Anti-CD272/BTLA antibody [EPR20539]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD272/BTLA antibody [EPR20539] (AB212089)

Immunofluorescent analysis of 100% methanol fixed A20 (mouse reticulum sarcoma cell line) cells labeling CD272/BTLA with ab212089 at 1/100 dilution, followed by by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and membranous staining on A20 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

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

• Flow Cyt

Lab

Flow Cytometry - Anti-CD272/BTLA antibody [EPR20539] (AB212089)

Flow cytometric analysis of A20 (mouse reticulum sarcoma cell line) cell line labeling CD272/BTLA with ab212089 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Total viable cells were gated for the FC image.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD272/BTLA antibody [EPR20539] (AB212089)

Flow cytometric analysis of mouse splenocytes labeling CD272/BTLA with ab212089 at 1/500 dilution (right) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Mouse splenocytes stained with CD3 and ab212089. Total viable cells were gated for the FC image. T cells (CD3+, upper population) show a moderate positive signal while B cells (lower right population in right panel) show stronger positive signal. The expression pattern is also reported in literature (PMID : 19949073).

• IP

Lab

Immunoprecipitation - Anti-CD272/BTLA antibody [EPR20539] (AB212089)

CD272/BTLA was immunoprecipitated from 0.35 mg mouse spleen lysate with ab212089 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab212089 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution

Lane 1 : Mouse spleen lysate 10 μg (Input).

Lane 2 : ab212089 IP in mouse spleen lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab212089 in mouse spleen lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-CD272/BTLA antibody [EPR20539] (ab212089)

Predicted band size: 33 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-CD272/BTLA antibody [EPR20539] (AB212089)

Exposure time : Lane 1 : 5 seconds; Lanes 2-3 : 30 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

Lane 1:

Western blot - Anti-CD272/BTLA antibody [EPR20539] (ab212089) at 1/10000 dilution

Lanes 2 - 3:

Western blot - Anti-CD272/BTLA antibody [EPR20539] (ab212089) at 1/1000 dilution

Lane 1:

A20 (mouse reticulum sarcoma cell line) whole cell lysate at 20 µg

Lane 2:

Mouse spleen lysate at 20 µg

Lane 3:

Mouse lymph node lysate at 20 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: 33 kDa

Observed band size: 55-70 kDa

true