Recombinant Anti-PU.1/Spi1 antibody [EPR25123-110] (ab302623)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR25123-110] to PU.1/Spi1
- Suitable for: IP, ChIP, ChIC/CUT&RUN-seq, WB, ICC/IF, Flow Cyt (Intra), IHC-P
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-PU.1/Spi1 antibody [EPR25123-110]
See all PU.1/Spi1 primary antibodies -
Description
Rabbit monoclonal [EPR25123-110] to PU.1/Spi1 -
Host species
Rabbit -
Tested applications
Suitable for: IP, ChIP, ChIC/CUT&RUN-seq, WB, ICC/IF, Flow Cyt (Intra), IHC-Pmore details -
Species reactivity
Reacts with: Human
Does not react with: Mouse, Rat -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: THP-1, U937, Daudi, HeLa whole cell lysates. IHC-P: Human colon and diffuse large B-cell lymphoma FFPE tissue sections. ICC/IF: THP-1 and U-937 cell lines. Flow Cyt (Intra): HeLa and U937 cells IP: THP-1 whole cell lysate. ChIP: U-937 cell line.
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General notes
This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR25123-110 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab302623 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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IP |
1/30.
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ChIP |
Use a concentration of 5 µg/ml.
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ChIC/CUT&RUN-seq |
Use at an assay dependent concentration.
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WB |
1/1000. Detects a band of approximately 31 kDa (predicted molecular weight: 31 kDa).
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ICC/IF |
1/250.
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Flow Cyt (Intra) |
1/500.
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IHC-P |
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Notes |
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IP
1/30. |
ChIP
Use a concentration of 5 µg/ml. |
ChIC/CUT&RUN-seq
Use at an assay dependent concentration. |
WB
1/1000. Detects a band of approximately 31 kDa (predicted molecular weight: 31 kDa). |
ICC/IF
1/250. |
Flow Cyt (Intra)
1/500. |
IHC-P
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Target
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Function
Binds to the PU-box, a purine-rich DNA sequence (5'-GAGGAA-3') that can act as a lymphoid-specific enhancer. This protein is a transcriptional activator that may be specifically involved in the differentiation or activation of macrophages or B-cells. Also binds RNA and may modulate pre-mRNA splicing. -
Sequence similarities
Belongs to the ETS family.
Contains 1 ETS DNA-binding domain. -
Cellular localization
Nucleus. - Information by UniProt
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Database links
- Entrez Gene: 6688 Human
- Omim: 165170 Human
- SwissProt: P17947 Human
- Unigene: 502511 Human
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Alternative names
- transcription factor spi1 antibody
- 31 kDa Transforming Protein antibody
- 31 kDa-transforming protein antibody
see all
Images
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ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/µL, 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.
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ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/µL, 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.
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ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/µL, 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.
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All lanes : Anti-PU.1/Spi1 antibody [EPR25123-110] (ab302623) at 1/1000 dilution
Lane 1 : THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lane 2 : U937 (human histiocytic lymphoma monocyte), whole cell lysate
Lane 3 : Daudi (human Burkitt's lymphoma lymphoblast), whole cell lysate
Lane 4 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 31 kDa
Observed band size: 31 kDa
Exposure time: 3 minutesBlocking / Diluting buffer and concentration: 5% NFDM/TBST
Negative control: HeLa (PMID: 27010793)
This blot was developed using a high sensitivity ECL substrate. -
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
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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
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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.
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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.
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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)
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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.
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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
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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
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (0)
ab302623 has not yet been referenced specifically in any publications.