Anti-SUFU antibody [EPR30547-60]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Wildtype HAP 1 (human chronic myelogenous leukemia near-haploid cell) and SUFU KO HAP1(SUFU knockout HAP1) cells labelling SUFU with ab325424 at 1/200 (2.445 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly cytoplasmic staining in wildtype HAP1 cells (shown in green), showing no staining in SUFU knockout HAP1 cells. 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 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Magenta).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Wildtype HEK-293T (human embryonic kidney epithelial cell), ab255449 and SUFU KO HEK-293T (SUFU knockout HEK-293T), ab267282 cells labelling SUFU with ab325424 at 1/200 (2.445 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly cytoplasmic staining in wildtype HEK-293T cells (shown in green), showing no staining in SUFU knockout HEK-293T cells. 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 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Magenta).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labeling SUFU with ab325424 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human cervical cancer.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling SUFU with ab325424 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human testis (PMID : 10564661).

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling SUFU with ab325424 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : Weak staining in human liver.

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunohistochemical analysis of paraffin-embedded Panel A : Wildtype HAP 1 (human chronic myelogenous leukemia near-haploid cell) and Panel B : SUFU KO HAP1 (SUFU knockout human chronic myelogenous leukemia near-haploid cell) tissue labeling SUFU with ab325424 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in wild-type HAP1 (Panel A), no staining in SUFU KO HAP1 (Panel B).

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunohistochemical analysis of paraffin-embedded Human prostatic hyperplasia tissue labeling SUFU with ab325424 at 1/500 (0.978 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human prostatic hyperplasia (PMID : 15482598).

The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SUFU antibody [EPR30547-60] (AB325424)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling SUFU with ab325424 at 1/200 (2.445 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly 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 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Magenta).

-ve control 1 : ab325424 at a 1/200 dilution followed by ab150120 at a 1/1000 dilution.

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

• WB

Lab

Western blot - Anti-SUFU antibody [EPR30547-60] (AB325424)

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

Performed under reducing conditions.

In Western blot, ab325424 was shown to bind specifically to SUFU. Target of interest was observed at 30, 35, 53 kDa in wild-type HAP1cell lysates (lane 1) and in wild-type HEK cell lysates (lane 3) with no signal observed at this size in SUFU knockout HAP1cell line (lane 2) and SUFU knockout HEK293T cell line (lane 4, knockout cell line ab267282 / knockout cell lysate ab257718).

The low MW band are due to degradation.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1 : 100000 (15Kda).

All lanes:

Western blot - Anti-SUFU antibody [EPR30547-60] (ab325424) at 1/1000 dilution

Lane 1:

Parental HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

SUFU knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Parental HEK293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

SUFU knockout HEK293T whole cell lysate at 20 µg

Lane 5:

HEL (human erythroleukemia erythroblast) whole cell lysate at 20 µg

Lane 6:

293T whole cell lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast) 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: 30 kDa,35 kDa,53 kDa,37 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-SUFU antibody [EPR30547-60] (AB325424)

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

Low expression : brain(PMID : 10564661)

The low MW band are due to degradation.

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

Exposure time : Lane 1 : 10 seconds, lanes 2-3 : 3 seconds

All lanes:

Western blot - Anti-SUFU antibody [EPR30547-60] (ab325424) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 30 kDa,35 kDa,53 kDa,36 kDa

false