Anti-SNX1 (phospho S188) antibody [EPR26023-114]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (AB314541)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / 293T cells transfected with a SNX1 expression vector containing a myc tag (Right) cells labelling SNX1 (phospho S188) with ab314541 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (AB314541)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (Human embryonic kidneyepithelial cell) transfected with a SNX1 expression vector containing a myc tag cells labelling SNX1 (phospho S188) with ab314541 at 1/50 (10.02 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/mL) dilution (Green). Confocal image showing cytoplasmic staining in 293T cells transfected with a human SNX1 expression vector containing a myc tag. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain at 1/100 (0.38ug/ ml) dilution (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 ug/mL) dilution.

• IP

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Immunoprecipitation - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (AB314541)

SNX1 (phospho S188) was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) cells transfected with a human SNX1 expression vector containing a His-tag, whole cell lysate with ab314541 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314541 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T (human embryonic kidney epithelial cell) cells transfected with a human SNX1 expression vector containing a His-tag, whole cell lysate

Lane 2 : ab314541 IP in 293T (human embryonic kidney epithelial cell) cells transfected with a human SNX1 expression vector containing a His-tag, whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314541 in 293T cells transfected with a human SNX1 expression vector containing a His-tag, whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (ab314541) at 1/30 dilution

All lanes:

293T (human embryonic kidney epithelial cell) cells transfected with a human SNX1 expression vector containing a His-tag, whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

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Western blot - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (AB314541)

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

All lanes:

Western blot - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (ab314541) at 1/1000 dilution

Lane 1:

Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

293T treated with 100 nM Calycin A for 30 minutes whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 4:

293T treated with 100 nM Calycin A for 30 minutes whole cell lysate (phosphatase treated membrane) at 20 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 1/20000 dilution

Observed band size: 74 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (AB314541)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (ab314541) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human SNX1 expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human SNX1(S188A mutant) expression vector containing a His-tag, whole cell lysate at 20 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

Observed band size: 74 kDa,36 kDa

false

Exposure time: 26s

• Dot

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Dot Blot - Anti-SNX1 (phospho S188) antibody [EPR26023-114] (AB314541)

Dot blot analysis of SNX1 (phospho S188) using ab314541 at 1 : 1000 (0.501 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Exposure time : 180 seconds

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