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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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 labelling PU.1/Spi1 with ab302623 at 1/250 (1.868 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/mL dilution (Green). Confocal image showing nuclear staining in THP-1 cell line.Negative control : Hela (PMID : 27010793) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/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 2ug/mL dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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 labelling PU.1/Spi1 with ab302623 at 1/500 dilution (0.1ug) (Red) (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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 labelling PU.1/Spi1 with ab302623 at 1/500 dilution (0.1ug) (Red) (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control : HeLa (PMID : 27010793)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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 labelling PU.1/Spi1 with ab302623 at 1/250 (1.868 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/mL dilution (Green). Confocal image showing nuclear staining in U-937 cell line. is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/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 2ug/mL dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

This data was developed using ab302623, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Human diffuse large tissue labeling PU.1/Spi1 with ab302623 at 1/1000 (0.467 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ChIP

Lab

ChIP - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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

• IP

Supplier Data

Immunoprecipitation - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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.

All lanes:

Immunoprecipitation - Anti-PU.1/Spi1 antibody [EPR25123-110] (<a href='/en-us/products/primary-antibodies/pu1-spi1-antibody-epr25123-110-ab302623'>ab302623</a>) at 1/30 dilution

All lanes:

THP-1 (human monocytic leukemia monocyte), 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

Predicted band size: 31 kDa

Observed band size: 31 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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

Exposure time : Lane 1-2 : 180 seconds; Lane 3-4 : 20 seconds

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

ab76543 is more sensitive than ab302623 in WB testing.

Lanes 1 - 2:

Western blot - Anti-PU.1/Spi1 antibody [EPR25123-110] (<a href='/en-us/products/primary-antibodies/pu1-spi1-antibody-epr25123-110-ab302623'>ab302623</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (ab302624) at 1/1000 dilution

Lanes 3 - 4:

Western blot - Anti-PU.1/Spi1 antibody [EPR3158Y] (<a href='/en-us/products/primary-antibodies/pu1-spi1-antibody-epr3158y-ab76543'>ab76543</a>) at 1/1000 dilution

Lanes 1 and 3:

Daudi whole cell lysate at 20 µg

Lanes 2 and 4:

Raji 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

Predicted band size: 31 kDa

Observed band size: 37-42 kDa

false

• WB

Supplier Data

Western blot - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

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

Blocking and 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.

All lanes:

Western blot - Anti-PU.1/Spi1 antibody [EPR25123-110] (<a href='/en-us/products/primary-antibodies/pu1-spi1-antibody-epr25123-110-ab302623'>ab302623</a>) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

Lane 2:

U937 (human histiocytic lymphoma monocyte), whole cell lysate at 20 µg

Lane 3:

Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma 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: 31 kDa

true

Exposure time: 3min

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

"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."

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

"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."

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-PU.1/Spi1 antibody [EPR25123-110] (BSA and Azide free) (AB302624)

"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."