Anti-VPS4a antibody [EPR30587-24]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VPS4a antibody [EPR30587-24] (AB327386)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Wildtype HAP 1 (human chronic myelogenous leukemia near-haploid cell) and VPS4A KO HAP1(VPS4A Knockout human chronic myelogenous leukemia near-haploid cell) labelling VPS4a with ab327386 at 1/1000 (0.511 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 wildtype HAP1 cells (shown in green), showing no staining VPS4A 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 (Magenta). The Nuclear counterstain was DAPI (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VPS4a antibody [EPR30587-24] (AB327386)

Immunohistochemical analysis of paraffin-embedded Human endomatrial cancer tissue labeling VPS4a with ab327386 at 1/1000 (0.511 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human endomatrial 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-VPS4a antibody [EPR30587-24] (AB327386)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling VPS4a with ab327386 at 1/1000 (0.511 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human kidney. 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-VPS4a antibody [EPR30587-24] (AB327386)

Immunohistochemical analysis of paraffin-embedded (A) : Wildtype HAP1(human chronic myelogenous leukemia near-haploid cell) cell pellet and (B) : VPS4A knockout HAP1 cell pellet labeling VPS4a with ab327386 at 1/1000 (0.511 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) Wildtype HAP1 cell pellet, no staining on (B) VPS4A knockout HAP1 cell pellet. 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-VPS4a antibody [EPR30587-24] (AB327386)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling VPS4a with ab327386 at 1/1000 (0.511 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human ovarian 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

• WB

Lab

Western blot - Anti-VPS4a antibody [EPR30587-24] (AB327386)

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

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

All lanes:

Western blot - Anti-VPS4a antibody [EPR30587-24] (ab327386) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Mouse liver tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse lung 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: 48 kDa

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-VPS4a antibody [EPR30587-24] (AB327386)

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

This antibody does not cross-react with recombinant human VPS4B by western blot.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(48KDa).

All lanes:

Western blot - Anti-VPS4a antibody [EPR30587-24] (ab327386) at 1/1000 dilution

Lane 1:

His-tagged human VPS4A recombinant protein at 10 ng

Lane 2:

His-tagged human VPS4B 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: 48 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-VPS4a antibody [EPR30587-24] (AB327386)

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

In Western blot, ab327386 was shown to bind specifically to VPS4A. Target of interest was observed at 48 kDa in wild-type HAP1 lysates (lane 1) with no signal observed at this size in VPS4A knockout cell line (lane 2) .

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

All lanes:

Western blot - Anti-VPS4a antibody [EPR30587-24] (ab327386) at 1/1000 dilution

Lane 1:

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

Lane 2:

VPS4A KO HAP1 whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 5:

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

Lane 6:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

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

Lane 8:

Neuro-2a (mouse neuroblastoma neuroblast) 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: 48 kDa,36 kDa

false

Exposure time: 180s