Anti-PU.1/Spi1 抗体 [EPR25123-110] (BSA and Azide free) (ab302624)

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

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

Negative control: HeLa (PMID: 27010793)
This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

This data was developed using the same antibody clone in a different buffer formulation (ab302623). 
ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 THP-1 (Human monocytic leukemia monocyte) cells and 5 µg of ab302623 [EPR25123-110]. 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.

This data was developed using the same antibody clone in a different buffer formulation (ab302623). 
ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 THP-1 (Human monocytic leukemia monocyte) cells and 5 µg of ab302623 [EPR25123-110]. 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.

This data was developed using the same antibody clone in a different buffer formulation (ab302623).
ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 THP-1 (Human monocytic leukemia monocyte) cells and 5 µg of ab302623 [EPR25123-110]. 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.

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling PU.1/Spi1 with ab302623 at 1/1000 (0.467 µg/ml) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit). Positive staining in immune cells of human colon (PMID: 28681454). The section was incubated with ab302623 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 Leica DS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human diffuse large B-cell lymphoma tissue labeling PU.1/Spi1 with ab302623 at 1/1000 (0.467 µg/ml) followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection kit). Positive staining in human diffuse large B-cell lymphoma (PMID:16648862). The section was incubated with ab302623 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 Leica DS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labeling PU.1/Spi1 with ab302623 at 1/250 (1.868 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2µg/ml) dilution (Green). Confocal image showing nuclear staining in THP-1 cell line. Negative control: Hela (PMID: 27010793). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (Red). 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 (2µg/ml) dilution.

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-937 (human histiocytic lymphoma monocyte) cells labeling PU.1/Spi1 with ab302623 at 1/250 (1.868 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2µg/ml) dilution (Green). Confocal image showing nuclear staining in U-937 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (Red). 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 (2µg/ml) dilution.

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell, Left) / U937 (human histiocytic lymphoma monocyte, Right) cells labeling PU.1/Spi1 with ab302623 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control: HeLa (PMID: 27010793)

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (human monocytic leukemia monocyte) cells labeling PU.1/Spi1 with ab302623 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

PU.1/Spi1 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte), whole cell lysate 10 µg with ab302623 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab302623 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (human monocytic leukemia monocyte), whole cell lysate 10 µg

Lane 2: ab302623 IP in THP-1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab302623 in THP-1 whole cell lysate

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

Observed MW(KDa): 31 kDa

Exposure time: 3 minutes

This data was developed using ab302623, the same antibody clone in a different buffer formulation.

Chromatin was prepared from U-937 cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min. The ChIP was performed with 25 µg of chromatin, 5 µg of ab302623 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

Primers and probes are from paper PMID:21402070, 21094529, 26622774