Anti-LAP2 alpha 抗体 [EPR28581-87]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Immunohistochemical analysis of paraffin-embedded Human gastric carcinoma tissue labeling LAP2 alpha with ab317028 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human gastric carcinoma (PMID : 22745766). The section was incubated with ab317028 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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized TMPO KO HEK293T (LAP2 alpha knock out human embryonic kidney epithelial cell, Left) / Parental HEK293T (Right) cells labelling LAP2 alpha with ab317028 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling LAP2 alpha with ab317028 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human tonsil. The section was incubated with ab317028 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

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Immunocytochemistry/ Immunofluorescence - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized TMPO KO HEK293T (TMPO knockout human embryonic kidney epithelial cell) (ab266774) cells labelling LAP2 alpha with ab317028 at 1/500 (1.046 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 nuclear staining in parental HEK293T cells (shown in green), no staining in TMPO KO HEK293T cells. The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Magenta). 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-LAP2 alpha antibody [EPR28581-87] (AB317028)

LAP2 alpha was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell) whole cell lysate with ab317028 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317028 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : ab317028 IP in HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317028 in HEK-293 whole cell lysate.

Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-LAP2 alpha antibody [EPR28581-87] (ab317028) at 1/30 dilution

All lanes:

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

Secondary

All lanes:

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

false

Exposure time: 180s

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling LAP2 alpha with ab317028 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling LAP2 alpha with ab317028 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen. The section was incubated with ab317028 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling LAP2 alpha with ab317028 at 1/2000 (0.262 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat spleen. The section was incubated with ab317028 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling LAP2 alpha with ab317028 at 1/500 (1.046 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 nuclear staining in NIH/3T3 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Magenta). 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.

• WB

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Western blot - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

In Western blot, ab317028 was shown to bind specifically to LAP2 alpha. Target of interest was observed at 76 kDa in wild-type HEK-293 cell lysates (lane 1/4) with no signal observed at this size in TMPO knockout cell line (lane 2/5) (knockout cell line ab266774 / knockout cell lysate ab257747).

In lanes 1-3, the lysates were stored at -80℃ prior to Western Blotting. The bands beneath the target band (76 kDa) are likey to be degradation products. In lanes 4-13, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

The identity of the higher MW band at approximately 250 kDa (in lane 10) is unknown.

The lanes 1-2 were developed using a high sensitivity ECL substrate.

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

Exposure time : Lanes 1-2 : 81 seconds, lanes 3-13 : 180 seconds

All lanes:

Western blot - Anti-LAP2 alpha antibody [EPR28581-87] (ab317028) at 1/1000 dilution

Lane 1:

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

Lanes 2 and 5:

TMPO knockout HEK-293 whole cell lysate at 20 µg

Lane 3:

HEL (erythroleukemia erythroblast) whole cell lysate at 20 µg

Lane 4:

Wild-type HEK-293 whole cell lysate at 20 µg

Lane 6:

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

Lane 7:

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

Lane 8:

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

Lane 9:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 10:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 11:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 12:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 13:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 76 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

The bands beneath the target band (76 kDa) are likely to be degraded target fragments.

All lanes:

Western blot - Anti-LAP2 alpha antibody [EPR28581-87] (ab317028) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Rat testis tissue lysate at 20 µg

Lane 4:

Rat spleen tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 76 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-LAP2 alpha antibody [EPR28581-87] (AB317028)

This antibody does not cross-react with human LAP2 beta, LAP2 gamma and LAP2 zeta.

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

All lanes:

Western blot - Anti-LAP2 alpha antibody [EPR28581-87] (ab317028) at 1/1000 dilution

Lane 1:

His-tagged human LAP2 alpha fragment at 100 ng

Lane 2:

His-tagged human LAP2 beta recombinant protein at 100 ng

Lane 3:

His-tagged human LAP2 gamma recombinant protein at 100 ng

Lane 4:

His-tagged human LAP2 zeta recombinant protein at 100 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 57 kDa

false

Exposure time: 180s