Overview

  • Product name

    Anti-SP1 antibody [EPR22648-50]
    See all SP1 primary antibodies
  • Description

    Rabbit monoclonal [EPR22648-50] to SP1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IP, ChIPmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment within Human SP1 aa 450-650. The exact sequence is proprietary.
    Database link: P08047

  • Positive control

    • WB: HAP1, HeLa, K-562 and HEK-293T whole cell lysate. IHC-P: Human breast carcinoma and gastric carcinoma tissue. ICC/IF: HeLa cells. Flow: HeLa cells. IP: HeLa cell lysate.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab231778 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
WB 1/1000. Predicted molecular weight: 81 kDa.
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

ICC/IF 1/100.
Flow Cyt 1/60.
IP 1/30.
ChIP Use at an assay dependent concentration.

Use 5 µg.

Target

  • Function

    Transcription factor that can activate or repress transcription in response to physiological and pathological stimuli. Binds with high affinity to GC-rich motifs and regulates the expression of a large number of genes involved in a variety of processes such as cell growth, apoptosis, differentiation and immune responses. Highly regulated by post-translational modifications (phosphorylations, sumoylation, proteolytic cleavage, glycosylation and acetylation). Binds also the PDGFR-alpha G-box promoter. May have a role in modulating the cellular response to DNA damage. Implicated in chromatin remodeling. Plays a role in the recruitment of SMARCA4/BRG1 on the c-FOS promoter. Plays an essential role in the regulation of FE65 gene expression. In complex with ATF7IP, maintains telomerase activity in cancer cells by inducing TERT and TERC gene expression.
  • Tissue specificity

    Up-regulated in adenocarcinomas of the stomach (at protein level).
  • Sequence similarities

    Belongs to the Sp1 C2H2-type zinc-finger protein family.
    Contains 3 C2H2-type zinc fingers.
  • Post-translational
    modifications

    Phosphorylated on multiple serine and threonine residues. Phosphorylation is coupled to ubiquitination, sumoylation and proteolytic processing. Phosphorylation on Ser-59 enhances proteolytic cleavage. Phosphorylation on Ser-7 enhances ubiquitination and protein degradation. Hyperphosphorylation on Ser-101 in response to DNA damage has no effect on transcriptional activity. MAPK1/MAPK3-mediated phosphorylation on Thr-453 and Thr-739 enhances VEGF transcription but, represses FGF2-triggered PDGFR-alpha transcription. Also implicated in the repression of RECK by ERBB2. Hyperphosphorylated on Thr-278 and Thr-739 during mitosis by MAPK8 shielding SP1 from degradation by the ubiquitin-dependent pathway. Phosphorylated in the zinc-finger domain by calmodulin-activated PKCzeta. Phosphorylation on Ser-641 by PKCzeta is critical for TSA-activated LHR gene expression through release of its repressor, p107. Phosphorylation on Thr-668, Ser-670 and Thr-681 is stimulated by angiotensin II via the AT1 receptor inducing increased binding to the PDGF-D promoter. This phosphorylation is increased in injured artey wall. Ser-59 and Thr-681 can both be dephosphorylated by PP2A during cell-cycle interphase. Dephosphorylation on Ser-59 leads to increased chromatin association during interphase and increases the transcriptional activity. On insulin stimulation, sequentially glycosylated and phosphorylated on several C-terminal serine and threonine residues.
    Acetylated. Acetylation/deacetylation events affect transcriptional activity. Deacetylation leads to an increase in the expression the 12(s)-lipooxygenase gene though recruitment of p300 to the promoter.
    Ubiquitinated. Ubiquitination occurs on the C-terminal proteolytically-cleaved peptide and is triggered by phosphorylation.
    Sumoylated by SUMO1. Sumoylation modulates proteolytic cleavage of the N-terminal repressor domain. Sumoylation levels are attenuated during tumorigenesis. Phosphorylation mediates SP1 desumoylation.
    Proteolytic cleavage in the N-terminal repressor domain is prevented by sumoylation. The C-terminal cleaved product is susceptible to degradation.
    O-glycosylated; contains at least 8 N-acetylglucosamine side chains. Levels are controlled by insulin and the SP1 phosphorylation states. Insulin-mediated O-glycosylation locates SP1 to the nucleus, where it is sequentially deglycosylated and phosphorylated. O-glycosylation affects transcriptional activity through disrupting the interaction with a number of transcription factors including ELF1 and NFYA. Also inhibits interaction with the HIV1 promoter. Inhibited by peroxisomome proliferator receptor gamma (PPARgamma).
  • Cellular localization

    Nucleus. Cytoplasm. Nuclear location is governed by glycosylated/phosphorylated states. Insulin promotes nuclear location, while glucagon favors cytoplasmic location.
  • Information by UniProt
  • Database links

  • Alternative names

    • SP 1 antibody
    • SP1 antibody
    • Sp1 transcription factor antibody
    • SP1_HUMAN antibody
    • Specificity protein 1 antibody
    • Transcription factor Sp1 antibody
    • TSFP 1 antibody
    • TSFP1 antibody
    see all

Images

  • All lanes : Anti-SP1 antibody [EPR22648-50] (ab231778) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : SP1 knockout HAP1 whole cell lysate
    Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
    Lane 4 : 293T (human embryonic kidney epithelial cell) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 81 kDa
    Observed band size: 98 kDa
    why is the actual band size different from the predicted?


    Exposure time: 8 seconds


    ab231778 was shown to specifically react with SP1 in wild-type HAP1 cells as signal was lost in SP1 knockout cells. Wild-type and SP1 knockout samples were subjected to SDS-PAGE. ab231778 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

     

    Degraded fragments (29-97kDa) of SP1 have been descried in the literature (PMID: 10329728).

  • SP1 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate using ab231778 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab231778 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

    Lane 1: HeLa whole cell lysate 10 µg (input).
    Lane 2: ab231778 IP in HeLa whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab231778 in HeLa whole cell lysate.

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

    Exposure time: 3 minutes.

  • Immunohistochemical analysis of paraffin-embedded human gastric carcinoma tissue labeling SP1 using ab231778 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counterstained with hematoxylin.

    Nuclear staining on tumor cells of human gastric carcinoma (PMID: 14695137). The section was incubated with ab231778 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). 

     

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling SP1 using ab231778 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counterstained with hematoxylin.

    Nuclear staining on tumor cells of human breast carcinoma (PMID: 14695137). The section was incubated with ab231778 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). 

     

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling SP1 (green) with ab231778 at 1/100 dilution, followed by an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Confocal image showing nuclear staining in HeLa cell line. The nuclear counterstain is DAPI (Blue). Tubulin was stained using an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (Red).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a AlexaFluor®488 Goat anti-Rabbit secondary (ab150077).

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SP1 using ab231778 at 1/60 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and a unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). The secondary antibody was a Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at a 1/2000 dilution.

  • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min.

    The ChIP was performed with 25 µg of chromatin, 5 µg of ab231778 (red), and 20 µl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach).

    Primers and probes are located in the first kb of the transcribed region.

References

ab231778 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

There are currently no Customer reviews or Questions for ab231778.
Please use the links above to contact us or submit feedback about this product.

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up