Anti-mNeonGreen antibody [EPR28835-76]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling mNeonGreen with ab321887 at 1/100 (4.98 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human cerebrum.
The section was incubated with ab321887 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a mNeonGreen-GPGPG-SP1 expression vector. (B) HEK-293T transfected with empty vector. tissue labeling mNeonGreen with ab321887 at 1/2000 (0.249 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) HEK-293T transfected with a mNeonGreen-GPGPG-SP1 expression vector. No staining on (B) HEK-293T transfected with empty vector.
The section was incubated with ab321887 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-mNeonGreen antibody [EPR28835-76] (AB321887)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling mNeonGreen with ab321887 at 1/2000 (0.249 ug/ml) dilution followed by ab150088 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150088) antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing positive staining in 293T cells transfected with a mNeonGreen-GPGPG-SP1 expression vector (shown in green). ab321887 is shown in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

was used to counterstain tubulin at 1/None dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150088 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150088) at 1/1000 2 ug/ml dilution.

• ChIP-seq

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ChIP-sequencing - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

Chromatin was prepared from Human SP1 knockout HeLa cell line (ab265519) cells overexpressing either mNeonGreen or mNeonGreen-SP1. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 μg ab321887 [EPR28835-76]. Assay quality control was conducted using 8 μg anti-SP1 (ab231778) on the same cell lines. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

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ChIP-sequencing - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

Chromatin was prepared from Human SP1 knockout HeLa cell line (ab265519) cells overexpressing either mNeonGreen or mNeonGreen-SP1. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 μg ab321887 [EPR28835-76]. Assay quality control was conducted using 8 μg anti-SP1 (ab231778) on the same cell lines. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

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ChIP-sequencing - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

Chromatin was prepared from Human SP1 knockout HeLa cell line (ab265519) cells overexpressing either mNeonGreen or mNeonGreen-SP1. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 μg ab321887 [EPR28835-76]. Assay quality control was conducted using 8 μg anti-SP1 (ab231778) on the same cell lines. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling mNeonGreen with ab321887 at 1/100 (4.98 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on rat cerebrum.
The section was incubated with ab321887 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-mNeonGreen antibody [EPR28835-76] (AB321887)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling mNeonGreen with ab321887 at 1/100 (4.98 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on mouse cerebrum.
The section was incubated with ab321887 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

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

ChIC/CUT&RUN was performed using pAG-MNase at a final concentration of 700 ng/mL. 2.5 x 10⁵ Human SP1 knockout HeLa cells (ab265519) over expressing either mNeonGreen or mNeonGreen-SP1 were used along with 5 μg of ab321887[EPR28835-76]. Assay quality control was conducted using 5 μg anti-SP1(ab231778) on the same cell lines. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

ChIC/CUT&RUN was performed using pAG-MNase at a final concentration of 700 ng/mL. 2.5 x 10⁵ Human SP1 knockout HeLa cells (ab265519) over expressing either mNeonGreen or mNeonGreen-SP1 were used along with 5 μg of ab321887[EPR28835-76]. Assay quality control was conducted using 5 μg anti-SP1(ab231778) on the same cell lines. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

ChIC/CUT&RUN was performed using pAG-MNase at a final concentration of 700 ng/mL. 2.5 x 10⁵ Human SP1 knockout HeLa cells (ab265519) over expressing either mNeonGreen or mNeonGreen-SP1 were used along with 5 μg of ab321887[EPR28835-76]. Assay quality control was conducted using 5 μg anti-SP1(ab231778) on the same cell lines. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• IP

Supplier Data

Immunoprecipitation - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

mNeonGreen was immunoprecipitated from 0.35 mg SP1 knockout HeLa cells transfected with a mNeonGreen-GPGPG-SP1 expression vector, whole cell lysate with ab321887 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab321887 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : SP1 knockout HeLa (human cervical adenocarcinoma epithelial) cells transfected with a mNeonGreen-GPGPG-SP1 expression vector, whole cell lysate
Lane 2 : ab321887 IP in SP1 knockout HeLa cells transfected with a mNeonGreen-GPGPG-SP1 expression vector, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab321887 in SP1 knockout HeLa cells transfected with a mNeonGreen-GPGPG-SP1 expression vector, whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-mNeonGreen antibody [EPR28835-76] (ab321887) at 1/30 dilution

All lanes:

SP1 knockout HeLa (human cervical adenocarcinoma epithelial) cells transfected with a mNeonGreen-GPGPG-SP1 expression vector, 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: 6s

• IP

Supplier Data

Immunoprecipitation - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

mNeonGreen was immunoprecipitated from 0.35 mg SP1 knockout HeLa cells transfected with a mNeonGreen expression vector containing a myc-His-tag®, whole cell lysate with ab321887 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab321887 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : SP1 knockout HeLa (human cervical adenocarcinoma epithelial) cells transfected with a mNeonGreen expression vector containing a myc-His-tag®, whole cell lysate 1ug
Lane 2 : ab321887 IP in SP1 knockout HeLa cells transfected with a mNeonGreen expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab321887 in SP1 knockout HeLa cells transfected with a mNeonGreen expression vector containing a myc-His-tag®, whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-mNeonGreen antibody [EPR28835-76] (ab321887) at 1/30 dilution

All lanes:

SP1 knockout HeLa (human cervical adenocarcinoma epithelial) cells transfected with a mNeonGreen expression vector containing a myc-His-tag&reg;, 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: 180s

• WB

Supplier Data

Western blot - Anti-mNeonGreen antibody [EPR28835-76] (AB321887)

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

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

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

In Western blot, Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-mNeonGreen antibody [EPR28835-76] (ab321887) at 1/1000 dilution

Lane 1:

SP1 knockout HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SP1 knockout HeLa cells transfected with a mNeonGreen-GPGPG-SP1 expression vector containing no His-tag&reg;, whole cell lysate at 20 µg

Lane 3:

SP1 knockout HeLa cells transfected with a mNeonGreen expression vector containing a myc-His-tag&reg;, 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 1/20000 dilution

Observed band size: 36 kDa,133 kDa,35 kDa

false

Exposure time: 81s