Anti-SNX2 antibody [EPR30535-531]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SNX2 antibody [EPR30535-531] (AB326422)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Parental HeLa (human cervical adenocarcinoma epithelial cell) and SNX2 KO HeLa, ab265908 cells labelling SNX2 with ab326422 at 1/100 (5.29 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in parental HeLa cells and no staining in SNX2 knockout HeLa cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SNX2 antibody [EPR30535-531] (AB326422)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling SNX2 with ab326422 at 1/50 (10.58 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in HeLa cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab326422 at 1/50 dilution, followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• IP

Lab

Immunoprecipitation - Anti-SNX2 antibody [EPR30535-531] (AB326422)

SNX2 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab326422 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab326422 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ab326422 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab326422 in HeLa whole cell lysate

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

Exposure time : 24 seconds.

All lanes:

Immunoprecipitation - Anti-SNX2 antibody [EPR30535-531] (ab326422) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab326422 at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab326422 in HeLa 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

Observed band size: 72 kDa

false

Exposure time: 24s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SNX2 antibody [EPR30535-531] (AB326422)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling SNX2 with ab326422 at 1/50 (10.58 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in NIH/3T3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab326422 at 1/50 dilution, followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• WB

Lab

Western blot - Anti-SNX2 antibody [EPR30535-531] (AB326422)

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

Performed under reducing conditions.

In Western blot, ab326422 was shown to bind specifically to SNX2. Target of interest was observed at 72 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in SNX2 knockout cell line (lane 2, knockout cell line ab265908 / knockout cell lysate ab259149).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-SNX2 antibody [EPR30535-531] (ab326422) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SNX2 knockout HeLa 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 1/20000 dilution

Observed band size: 72 kDa,36 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-SNX2 antibody [EPR30535-531] (AB326422)

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

This antibody does not cross-react with recombinant human SNX1 by western blot.

In Western blot, Anti-6X His tag® antibody [AD1.1.10] - (ab15149) (1 : 1000) (60KDa).

All lanes:

Western blot - Anti-SNX2 antibody [EPR30535-531] (ab326422) at 1/1000 dilution

Lane 1:

His-tagged human SNX2 fragment at 10 ng

Lane 2:

His-tagged human SNX1 recombinant protein at 10 ng

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: 60 kDa

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-SNX2 antibody [EPR30535-531] (AB326422)

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

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lane 1 : 26 seconds; Lanes 2-5 : 15 seconds.

All lanes:

Western blot - Anti-SNX2 antibody [EPR30535-531] (ab326422) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Mouse testis tissue lysate at 20 µg

Lane 5:

Mouse spleen tissue 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 1/20000 dilution

Observed band size: 72 kDa

false