Anti-XCL1+XCL2 抗体 [EPR26181-30] (BSA and Azide free) (ab302523)

All lanes : Anti-XCL1+XCL2 antibody [EPR26181-30] (ab302522) at 1/500 dilution

Lane 1 : Untreated HuT-78 (human Sezary syndrome cutaneous T lymphocyte), whole cell lysate
Lane 2 : HuT-78 treated with 80 nM TPA and 3 uM Ionomycin for 5h, 300 ng/ml BFA was then added for additional 4h, whole cell lysate

Lysates/proteins at 40 µg per lane.

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

Predicted band size: 12 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

The data was developed using ab302522, the same antibody clone in different buffer formulation. 

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

The expression of XCL1 are upregulated in response to TPA and Ionomycin treatment (PMID:29474842, 32948687).

All lanes : Anti-XCL1+XCL2 antibody [EPR26181-30] (ab302522) at 1/500 dilution

Lane 1 : Human XCL1 recombinant protein 10 ng
Lane 2 : Human XCL2 recombinant protein 10 ng

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

Predicted band size: 12 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

The data was developed using ab302522, the same antibody clone in different buffer formulation. 

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

This data was developed using ab302522, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labeling XCL1+XCL2 with ab302522 at 1/100 dilution (4.32 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing cytoplasmic staining in HEK-293T cells transfected with a human lymphotactin expression vector containing a myc-tag. Myc-Tag Mouse mAb (Alexa Fluor^® 647) was used to counterstain tubulin at 1/100 dilution (0.38 µg/ml) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

This data was developed using ab302522, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HuT-78 (human Sezary syndrome cutaneous T lymphocyte) cells labeling XCL1+XCL2 with ab302522 at 1/1000 dilution (0.432 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing increased cytoplasmic staining in HuT-78 cells treated with TPA (80 nM) and Ionomycin (3 uM) for 5 h, then add Brefeldin A (300ng/ml) for another 4 h. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/ml) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 2 µg/ml dilution.

This data was developed using ab302522, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HuT-78 (hµMan Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 3µM Ionomycin for 5h, then add 300 ng/ml BFA for another 4h (Red). Untreated control (Green) cells labeling XCL1+XCL2 with ab302522 at 1/500 dilution (0.1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

This data was developed using ab302522, the same antibody clone in a different buffer formulation.

Intracellular flow cytometric analysis of 2% paraformaldehyde fixed, 0.1% Tween-20 permeabilized human peripheral blood mononuclear cell (PBMC) treated with 80nM TPA, 1.3µM Ionomycin, 10µM BFA and 2µM Monensin for 6h (Right) / Untreated control (Left) cells labeling XCL1+XCL2 with ab302522 at 1/500 dilution (0.1 µg) (Right) compared with a isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Left).  A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

This data was developed using ab302522, the same antibody clone in a different buffer formulation.

XCL1+XCL2 was immunoprecipitated from 0.35 mg HuT-78 (human Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 3uM Ionomycin for 5h, 300ng/ml BFA was then added for additional 4h, whole cell lysate with ab302522 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab302522 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HuT-78 (human Sezary syndrome cutaneous T lymphocyte) treated with 80nM TPA and 3uM Ionomycin for 5h, 300ng/ml BFA was then added for additional 4h, whole cell lysate 10 µg (Inset)

Lane 2: ab302522 IP in HuT-78 whole cell lysate 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302522 in HuT-78 whole cell lysate

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

Exposure time: 10 seconds

Observed M_W (kDa): 15.