Anti-SLP-2 antibody [EPR18718] (ab191883) at 1/1000 dilution + Human fetal kidney lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 39 kDa


Exposure time: 1 minute

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-SLP-2 antibody [EPR18718] (ab191883) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : A549 (Human lung carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 39 kDa
Observed band size: 39 kDa


Exposure time: 30 seconds

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-SLP-2 antibody [EPR18718] (ab191883) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse spleen lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : Rat brain lysate
Lane 6 : NIH/3T3 (Mouse embryonic fibroblast cell line) 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: 39 kDa
Observed band size: 39 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

Exposure time: Lane1,2,3 and 4:3 minutes;Lane 5 and 6:1 minute.

Immunofluorescence staining of HeLa cells with ab191883 at a working dilution of 1 in 250, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1 in 1000 (shown in green). The mitochondria were stained with ab33985 (mouse monoclonal to COX-IV, mitochondrial marker) at 1/1000 and ab150120 at 1/1000 (shown in red). The cells were fixed in 100% methanol and permeabilized using 0.1% triton-X 100. The negative controls are shown in bottom panels. For negative control 1, ab191883 was used at a dilution of 1/250 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/1000. For negative control 2, ab7291 (mouse monoclonal to alpha tubulin) was used at a dilution of 1/1000 followed by ab150077 (Alexa Fluor^® 488 goat anti-rabbit) at a dilution of 1/1000.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling SLP-2 with ab191883 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin -Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).
The negative controls are as follows:-
-ve control 1: ab191883 at 1/250 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized Jurkat cells (Human T cell leukemia cell line from peripheral blood) labeling SLP-2 with ab191883 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on Jurkat cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin -Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).
The negative controls are as follows:-
-ve control 1: ab191883 at 1/250 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling SLP-2 with ab191883 at 1/120 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.

SLP-2 was immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate with ab191883 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab191883 at 1/1000 dilution. Veriblot for IP (HRP) (ab13136) was used as secondary antibody at 1/10000 dilution.
Lane 1: Jurkat whole cell lysate 10µg (Input).
Lane 2: ab191883 IP in Jurkat whole cell lysate.
Lane 3: Rabbit IgG,monoclonal [EPR25A] (ab172730) instead of ab191883 in Jurkat whole cell cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.