Anti-SLP-2 antibody [EP18708]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SLP-2 antibody [EP18708] (AB191884)

Intracellular Flow Cytometry analysis of Jurkat cells labelling SLP-2 (red) with purified ab191884 at dilution of 1/900. The secondary antibody used was Alexa Fluorr^® 488 goat-anti-rabbit IgG (1/2000). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Isotype control antibody used was Rabbit Monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-SLP-2 antibody [EP18708] (AB191884)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling SLP-2 with ab191884 at 1/500 dilution followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasm staining on Jurkat cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab191884 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-SLP-2 antibody [EP18708] (AB191884)

SLP-2 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab191884 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab191884 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate,10ug (Input).

Lane 2 : ab191884 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab191884 in HeLa whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-SLP-2 antibody [EP18708] (ab191884)

Predicted band size: 39 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-SLP-2 antibody [EP18708] (AB191884)

SLP-2 was immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate with ab191884 at 1/60 dilution. Western blot was performed from the immunoprecipitate using ab191884 at 1/1000 dilution (Panel A) or another commercial anti-SLP-2 antibody at 1/1000 dilution (Panel B). VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

IP application of ab191884, panel (A), was further validated by performing cross IP with an anti-SLP-2 antibody from another supplier, panel (B).

Lane 1 and 4 : Jurkat whole cell lysate 10ug (Input).

Lane 2 and 5 : ab191884 IP in Jurkat whole cell lysate.

Lane 3 and 6 : Rabbit monoclonal IgG (ab172730) instead of ab191884 in Jurkat whole cell lysate.

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

Exposure time : 30 seconds (Panels A and B).

All lanes:

Immunoprecipitation - Anti-SLP-2 antibody [EP18708] (ab191884)

Predicted band size: 39 kDa

Observed band size: 39 kDa

false

• WB

Lab

Western blot - Anti-SLP-2 antibody [EP18708] (AB191884)

Western blot : Anti-SLP-2 antibody [EP18708] ab191884 staining at 1/2000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 40 kDa in Wild-type A549 cell lysates with no signal observed at this size in STOML2 knockout A549 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

Lanes 1 - 4:

Western blot - Anti-SLP-2 antibody [EP18708] (ab191884) at 1/2000 dilution

Lanes 1 - 4:

Western blot - Anti-SLP-2 antibody [EP18708] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/slp-2-antibody-ep18708-bsa-and-azide-free-ab251098'>ab251098</a>) at 1/2000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

STOML2 knockout A549 at 20 µg

Lane 3:

HeLa at 20 µg

Lane 4:

PC-3 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 40 kDa

Observed band size: 40 kDa

false

• WB

Supplier Data

Western blot - Anti-SLP-2 antibody [EP18708] (AB191884)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SLP-2 antibody [EP18708] (ab191884) at 1/2000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-SLP-2 antibody [EP18708] (AB191884)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SLP-2 antibody [EP18708] (ab191884) at 1/2000 dilution

Lane 1:

Human adenocarcinoma of lung tissue lysate at 10 µg

Lane 2:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 10 µg

Lane 3:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-SLP-2 antibody [EP18708] (AB191884)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time Lanes 1-4 : 15 seconds; Lanes 5-7 : 5 seconds.

All lanes:

Western blot - Anti-SLP-2 antibody [EP18708] (ab191884) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Mouse kidney lysate at 10 µg

Lane 4:

Mouse spleen lysate at 10 µg

Lane 5:

Rat brain lysate at 10 µg

Lane 6:

Rat heart lysate at 10 µg

Lane 7:

Rat spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

false

• WB

Supplier Data

Western blot - Anti-SLP-2 antibody [EP18708] (AB191884)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SLP-2 antibody [EP18708] (ab191884) at 1/2000 dilution

Lane 1:

C6 (Rat glial tumor cells) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

false

Exposure time: 3s