Anti-Zfp281/ZNF281 antibody [EPR30593-518]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Zfp281/ZNF281 with ab325790 at 1/100 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human tonsil.

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-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling Zfp281/ZNF281 with ab325790 at 1/100 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in 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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling Zfp281/ZNF281 with ab325790 at 1/50 (10.48 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 nuclear staining in 293T 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 (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325790 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-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Zfp281/ZNF281 was immunoprecipitated from 0.35 mg U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate with ab325790 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325790 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate
Lane 2 : ab325790 IP in U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325790 in U-2 OS whole cell lysate

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

Exposure time : 41 seconds.

To minimize protein degradation, lysates were made fresh.

All lanes:

Immunoprecipitation - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (ab325790) at 1/1000 dilution

Lane 1:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab325790 at 1/30 IP in U-2 OS (human bone osteosarcoma 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 ab325790 in U-2 OS 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: 100 kDa

false

Exposure time: 41s

• ChIP-seq

Lab

ChIP-sequencing - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Chromatin was prepared from U-2 OS cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325790 [EPR30593-518]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Lab

ChIP-sequencing - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Chromatin was prepared from U-2 OS cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325790 [EPR30593-518]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Lab

ChIP-sequencing - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Chromatin was prepared from U-2 OS cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325790 [EPR30593-518]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Immunohistochemical analysis of paraffin-embedded Mouse breast cancer tissue labeling Zfp281/ZNF281 with ab325790 at 1/100 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse breast 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Zfp281/ZNF281 with ab325790 at 1/100 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in mouse 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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling Zfp281/ZNF281 with ab325790 at 1/50 (10.48 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 nuclear 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 (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325790 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.

• ChIP-seq

Lab

ChIP-sequencing - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Chromatin was prepared from NIH/3T3 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325790 [EPR30593-518]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IP

Lab

Immunoprecipitation - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Zfp281/ZNF281 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab325790 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325790 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 : ab325790 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325790 in NIH/3T3 whole cell lysate

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

Exposure time : 58 seconds.

To minimize protein degradation, lysates were made fresh.

All lanes:

Immunoprecipitation - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (ab325790) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

ab325790 at 1/30 IP in NIH/3T3 (mouse embryonic fibroblast) 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 ab325790 in NIH/3T3 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: 100 kDa

false

Exposure time: 58s

• ChIP-seq

Lab

ChIP-sequencing - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Chromatin was prepared from NIH/3T3 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325790 [EPR30593-518]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Lab

ChIP-sequencing - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

Chromatin was prepared from NIH/3T3 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325790 [EPR30593-518]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• WB

Lab

Western blot - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

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

To minimize protein degradation, lysates were made fresh.

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

Exposure time : Lanes 1-2 : 180 seconds, lanes 3-4 : 180 seconds

All lanes:

Western blot - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (ab325790) at 1/1000 dilution

Lane 1:

U-2 OS (human bone osteosarcoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

U-2 OS transfected with siRNA specifically targeting ZNF281 whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

293T (human embryonic kidney epithelial cell) 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: 100 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (AB325790)

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

To minimize protein degradation, lysates were made fresh.

Lanes 1-2 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 and Lane 3 is incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000.

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

All lanes:

Western blot - Anti-Zfp281/ZNF281 antibody [EPR30593-518] (ab325790) at 1/1000 dilution

Lane 1:

Mouse liver tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

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

Secondary

Lanes 1 - 2:

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

Lane 3:

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: 100 kDa,36 kDa

false

Exposure time: 180s