Anti-LDB1 antibody [EPR28912-75]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LDB1 antibody [EPR28912-75] (AB318978)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling LDB1 with ab318978 at 1/50 (9.9 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 mainly nuclear staining in Hela cell line (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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-LDB1 antibody [EPR28912-75] (AB318978)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling LDB1 with ab318978 at 1/50 dilution (1 ug)/magenta compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

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

• IP

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Immunoprecipitation - Anti-LDB1 antibody [EPR28912-75] (AB318978)

LDB1 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab318978 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318978 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 : ab318978 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab318978 in HeLa whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-LDB1 antibody [EPR28912-75] (ab318978) at 1/30 dilution

All lanes:

HeLa (human cervical adenocarcinoma epithelial cell) 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: 67s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LDB1 antibody [EPR28912-75] (AB318978)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling LDB1 with ab318978 at 1/50 (9.9 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 mainly nuclear staining in NIH/3T3 cell line (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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-LDB1 antibody [EPR28912-75] (AB318978)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling LDB1 with ab318978 at 1/50 dilution (1 ug)/magenta compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

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-LDB1 antibody [EPR28912-75] (AB318978)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling LDB1 with ab318978 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum (PMID : 30873428).
The section was incubated with ab318978 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LDB1 antibody [EPR28912-75] (AB318978)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling LDB1 with ab318978 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat cerebrum.
The section was incubated with ab318978 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-LDB1 antibody [EPR28912-75] (AB318978)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling LDB1 with ab318978 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen.
The section was incubated with ab318978 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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

• IP

Supplier Data

Immunoprecipitation - Anti-LDB1 antibody [EPR28912-75] (AB318978)

LDB1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab318978 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318978 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 2 : ab318978 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab318978 in NIH/3T3 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-LDB1 antibody [EPR28912-75] (ab318978) at 1/30 dilution

All lanes:

NIH/3T3 (mouse embryonic fibroblast) 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: 67s

• WB

Supplier Data

Western blot - Anti-LDB1 antibody [EPR28912-75] (AB318978)

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

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

All lanes:

Western blot - Anti-LDB1 antibody [EPR28912-75] (ab318978) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 10 µg

Lane 2:

HeLa transfected with siRNA specifically targeting LDB1 whole cell lysate 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: 46 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-LDB1 antibody [EPR28912-75] (AB318978)

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

The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

Exposure time : Lanes 1-3 : 59 seconds; Lanes 4-11 : 180 seconds

All lanes:

Western blot - Anti-LDB1 antibody [EPR28912-75] (ab318978) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Lane 4:

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

Lane 5:

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

Lane 6:

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

Lane 7:

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

Lane 8:

Mouse brain tissue lysate at 20 µg

Lane 9:

Mouse spleen tissue lysate at 20 µg

Lane 10:

Rat brain tissue lysate at 20 µg

Lane 11:

Rat 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 1/100000 dilution

Observed band size: 46 kDa

false

• WB

Supplier Data

Western blot - Anti-LDB1 antibody [EPR28912-75] (AB318978)

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

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

All lanes:

Western blot - Anti-LDB1 antibody [EPR28912-75] (ab318978) at 1/1000 dilution

Lane 1:

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

Lane 2:

K-562 (human chronic myelogenous leukemia lymphoblast) 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: 46 kDa,36 kDa

false

Exposure time: 180s

• Dot

Supplier Data

Dot Blot - Anti-LDB1 antibody [EPR28912-75] (AB318978)

Dot blot analysis of LDB1 using ab318978 at 1 : 1000 (0.495 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : Human LDB1 peptide
Lane2 : His-tagged human LDB2 recombinant protein

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

This antibody has weak cross reaction with human LDB2 by Dot.

All lanes:

Dot Blot - Anti-LDB1 antibody [EPR28912-75] (ab318978) at 1/1000 dilution

Lane 1:

Human LDB1 peptide

Lane 2:

His-tagged human LDB2 recombinant protein

Secondary

All lanes:

Dot 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

false

Exposure time: 180s