Anti-SNX17 (phospho S336) antibody [EPR26024-4]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SNX17 (phospho S336) antibody [EPR26024-4] (AB316103)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (Human embryonic kidneyepithelial cell) cells labelling SNX17 (phospho S336) with ab316103 at 1/1000 (0.506 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 SNX17 expression vector containing a myc tag, and no staining in 293T cells transfected with a human SNX17 (S336A mutant) expression vector containing a myc tag.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain at 1/100 (5 ug/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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SNX17 (phospho S336) antibody [EPR26024-4] (AB316103)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Upper Left) / 293T cells transfected with a human SNX17 expression vector containing a myc tag (Upper Right) / 293T cells transfected with a human SNX17 S336A mutant expression vector containing a myc tag (Lower Left) / 293T cells transfected with an empty expression vector containing a myc tag (Lower Right) cells labelling SNX17 (phospho S336) with ab316103 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Cells are co-stained with Myc tag conjugated to Alexa Fluor®647.

• IP

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Immunoprecipitation - Anti-SNX17 (phospho S336) antibody [EPR26024-4] (AB316103)

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

SNX17 (phospho S336) was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) cells transfected with a human SNX17 expression vector containing a His-tag, whole cell lysate with ab316103 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316103 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 SNX17 expression vector containing a His-tag, whole cell lysate
Lane 2 : ab316103 IP in 293T (human embryonic kidney epithelial cell) cells transfected with a human SNX17 expression vector containing a His-tag, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316103 in 293T cells transfected with a human SNX17 expression vector containing a His-tag, whole cell lysate.

All lanes:

Immunoprecipitation - Anti-SNX17 (phospho S336) antibody [EPR26024-4] (ab316103) at 1/30 dilution

All lanes:

293T (human embryonic kidney epithelial cell) cells transfected with a human SNX17 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: 10s

• WB

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Western blot - Anti-SNX17 (phospho S336) antibody [EPR26024-4] (AB316103)

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-SNX17 (phospho S336) antibody [EPR26024-4] (ab316103) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a His-tag, whole cell lysate (untreated membrane) at 10 µg

Lane 2:

293T cells transfected with a Human wild-type SNX17 expression vector containing a His-tag, whole cell lysate (untreated membrane) at 10 µg

Lane 3:

293T cells transfected with a Human SNX17 (S336A mutant) expression vector containing a His-tag, whole cell lysate (untreated membrane) at 10 µg

Lane 4:

293T cells transfected with an empty vector containing a His-tag, whole cell lysate (alkaline phosphatase treated membrane) at 10 µg

Lane 5:

293T cells transfected with a Human wild-type SNX17 expression vector containing a His-tag, whole cell lysate (alkaline phosphatase treated membrane) at 10 µg

Lane 6:

293T cells transfected with a Human SNX17 (S336A mutant) expression vector containing a His-tag, whole cell lysate (alkaline phosphatase treated membrane) 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

Observed band size: 55 kDa,36 kDa

false

Exposure time: 26s

• Dot

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Dot Blot - Anti-SNX17 (phospho S336) antibody [EPR26024-4] (AB316103)

Dot blot analysis of SNX17 (phospho S336) using ab316103 at 1 : 1000 (0.506 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.