SP3 Antibody
Description
Applications of SP3 Antibody
SP3 antibodies are widely used in biomedical research:
Role in Cancer Biology
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SP3 overexpression induces apoptosis through caspase-3/6-mediated cleavage, generating 60 kDa and 35 kDa fragments .
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In HER2-positive breast cancer, SP3 antibody demonstrates superior specificity compared to CB11 (86.9% agreement with CISH) :
| Parameter | SP3 Antibody | CB11 Antibody |
|---|---|---|
| Specificity | 99.1% | 98% |
| Sensitivity | 52.3% | 52% |
| PPV | 96% | 92% |
Diagnostic Utility
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SP3 antibody correlates strongly with HER2 gene amplification (23/24 SP3 3+ cases showed amplification) .
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Reduces equivocal HER2 2+ classifications compared to polyclonal antibodies .
Mechanistic Insights
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Post-Translational Regulation: Sumoylation at Lys-551 modulates SP3's nuclear localization and transcriptional activity .
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Apoptosis Link: Cleaved SP3 fragments (CP1/CP2) correlate with caspase activation in resistant cancer clones .
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Gene Regulation: Competes with SP1 to control genes involved in cell cycle, apoptosis, and angiogenesis .
Limitations and Considerations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Knockdown of SP3 in cells with non-risk alleles revealed upregulation of the nearby cyclin-dependent kinase inhibitor 1C (CDKN1C) gene. This suggests that CDKN1C might be a functional target of SNP rs163184, which modulates the binding activity of the locus for Sp3 and Lsd1/Kdm1a. PMID: 29207083
- Elevated SP3 expression has been linked to hepatocellular carcinoma. PMID: 28844709
- We established a new regulatory model of COL1A1 regulation by HIF-1, highlighting its relationship with the Sp3 transcription factor. These findings offer insights into the mechanisms controlling COL1A1 gene expression. PMID: 27521280
- Data indicate that the antitumor activity of trefoil factor 2 (TFF2) is mediated by an interaction with the transcription factor Sp3 in gastric cancer cells. PMID: 27668303
- Studies have shown that a 186bp region is the minimal essential region and that Sp3-GC1 binding is crucial for the basal expression of KLF5. PMID: 27940107
- Sp3 has been identified as a major regulator of BNIP3 in prostate cancer. PMID: 26012884
- Chromosome 2q31.1 is located between the genes encoding the Sp3 transcription factor, which regulates the expression of genes associated with glomerular function and the pathogenesis of nephropathy, and the CDCA7 transcription factor. PMID: 24029427
- SP3 interacts with the TNIP1 promoter and contributes to its basal and inducible expression. PMID: 23464785
- Findings suggest a role for the transcription factor Sp3 in sarcomas as a driver for the expression of the metastasis-related gene AFAP1L1 (actin filament-associated protein 1-like 1). PMID: 23326307
- The transcription factor Sp3 acts to reduce the expression of numerous genes with Sp3 binding sites in their promoters by inhibiting the transition of paused RNA PolII to productive elongation. PMID: 23401853
- Transcriptional control of the multi-drug transporter ABCB1 by the transcription factor Sp3 in various human tissues has been observed. PMID: 23133566
- AP-1 and Sp3 are key regulators of IL-1beta-mediated modulation of xylosyltransferase I expression. PMID: 23223231
- This study indicated that Sp3 was a major regulator of TFF2 expression. PMID: 23000412
- Sp1 or Sp3 siRNA knockdown reduced 1alpha,25-dihydroxyvitamin D3-regulated hCYP24A1 promoter activity. PMID: 22871965
- Extracellular signal-regulated kinase mitogen-activated protein kinase-dependent SOCS-3 gene induction requires c-Jun, signal transducer and activator of transcription 3, and specificity protein 3 transcription factors. PMID: 22311708
- The two factors Klf4 and Sp3 exert an overlapping repressor function through their binding to the Notch1 promoter. PMID: 20442780
- Results suggest that Sp3 plays a key role in the expression of NOX4 in various human cell lineages. PMID: 21235713
- Transcription of the transforming growth factor beta activating integrin beta8 subunit is regulated by SP3, AP-1, and the p38 pathway. PMID: 20519498
- The human proximal FOXP3 promoter is controlled by activation through the TCR involving PKC and the NF-kappaB subunit p65 and by inhibition through a negative feedback loop and SP3. PMID: 20462637
- Sp3-mediated transcriptional repression is at least partially attributed to competition for promoter-specific transcription factors. PMID: 11773047
- Findings suggest that Sp1 and Sp3 associate with the hTERT promoter, recruiting HDAC for the localized deacetylation of nucleosomal histones and transcriptional silencing of the hTERT gene in normal human somatic cells. PMID: 12151407
- CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities, thereby influencing the dynamic chromatin remodeling of estrogen-regulated genes. PMID: 12176973
- This protein plays a role in identifying regulatory elements in the human adipose most abundant gene transcript-1(apM-1) promoter. PMID: 12378384
- 5' UTR of Sp3 transcription factor PMID: 12411611
- This protein and Sp1 are involved in the upregulation of human deoxyribonuclease II transcription during the differentiation of HL-60 cells. PMID: 12694199
- Acetylation serves as a switch that controls the repressor and activator role of Sp3. PMID: 12837748
- TFIIB interacts with SP1/SP3 at the SP1 site. PMID: 12972613
- In conclusion, evidence indicates that AML-1, PU.1, and Sp3 cooperatively and directly mediate BPI-expression during myeloid differentiation. PMID: 14623259
- SP3 acts as a positive regulator on the core promoter of the human ZPK gene. PMID: 14697235
- S-nitrosoglutathione enhances Sp3 binding to DNA and transcription of CFTR at physiological concentrations, but inhibits Sp3 binding and CFTR transcription at nitrosative stress levels. PMID: 14766015
- An SP3 binding site in the IGFBP4 gene was identified, and the role of SP3 in regulating its promoter activity in CaCo-2 cells was investigated. PMID: 14767471
- Sp3 isoform ratios and activity are controlled at the translational level, which regulates the expression of genes during mitosis and has implications for cell cycle regulation and tumorigenesis. PMID: 14767558
- Sp3 binding is regulated by methylation in the core-promoter region of the chondromodulin-I gene. PMID: 15107420
- Four isoforms derive from alternative translational start sites at positions 1, 37, 856, and 907; the transcriptional activity of all the Sp3 isoforms is regulated by SUMO modification. PMID: 15247228
- Sp3 isoforms are sumoylated in vivo, and this post-translational modification plays a significant role in the regulation of Sp3-mediated transcription. PMID: 15494207
- SP3 protein negatively regulates beta myosin heavy chain gene expression during skeletal muscle inactivity. PMID: 15572681
- Ten genes were down-regulated following treatment of the T-ALL cells with 0.15 and 1.5 microg/mL of metal ores at 72 h. DNA-binding transcriptional activator activity. PMID: 15747776
- Sp3 is a potent activator of dopamine transporter transcriptional activity. PMID: 15816870
- Cytochrome b5 gene transcription is regulated by Sp3, GATA-6, and steroidogenic factor 1 in human adrenal NCI-H295A cells. PMID: 15831526
- Results highlight the important roles of Sp1, Sp3, and NF-Y in the transcription regulation of the Sp3 proximal promoter. PMID: 16024108
- The Sp3 D-domain modulates its protein levels and activation of the p21(CIP1/WAF1) promoter. PMID: 16081043
- Increased pAPCP promoter activity in the MCF10A cell line in response to DMBA treatment is mediated by Sp3. PMID: 16150893
- Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2. PMID: 16574784
- PADI3 expression is driven by Sp3 binding to the promoter region. PMID: 16671893
- Sp3 and Sp1 transcription factors play a crucial role in the expression of the Protein S gene. PMID: 16672217
- Sp3 binds to the gamma-ENaC promoter, and Sp3 binding is enhanced by butyrate. PMID: 17241874
- Data show that increased generation of C18-ceramide by hCerS1 expression mediates the association and recruitment of deacetylated Sp3/HDAC1 to the hTERT promoter, resulting in local histone H3 deacetylation and repression of the promoter. PMID: 17548428
- IL-1beta decreases type II TGFbeta receptor expression by inducing Sp3 via NFkappaB. PMID: 18053089
- Results demonstrate activation of the Sp3 proximal promoter upon overexpression of NF-1, c-Myb, B-Myb, c-Jun, and c-Fos, and repression after overexpression of E2F/DP1. PMID: 18342022
- Observations suggest that deficient expression of the SP3 gene occurs in Chinese MS patients, and that SP3 expression may correlate with the clinical manifestations of MS and play a role in its immunological pathogenesis. PMID: 18393243
Q&A
What is SP3 antibody and what epitopes does it recognize?
SP3 antibody exists in two main forms with distinct applications. The first type (26584-1-AP) is a rabbit polyclonal antibody targeting the SP3 transcription factor, a 781 amino acid protein containing three C2H2-type zinc fingers that belongs to the Sp1 C2H2-type zinc-finger protein family . The second type is a rabbit monoclonal antibody directed against the extracellular domain of the HER2 receptor, primarily used in breast cancer diagnosis and research .
SP3 transcription factor antibody has been validated in multiple experimental systems, particularly for Western blotting applications in PC-3 cells, C2C12 cells, and COLO 320 cells . For HER2 receptor detection, SP3 antibody offers distinct advantages in membrane protein visualization compared to other commercially available antibodies.
What are the species reactivity profiles for SP3 antibody?
According to validated product information, the SP3 antibody targeting the transcription factor (26584-1-AP) demonstrates confirmed reactivity with human and mouse samples . When designing experimental protocols, researchers should consider this species compatibility to ensure appropriate antibody-antigen binding specificity. Cross-reactivity testing with additional species may be required for applications beyond the validated reactivity profile.
What are the recommended dilution parameters for SP3 antibody applications?
For SP3 antibody targeting the transcription factor (26584-1-AP), the following dilution ranges have been experimentally validated:
| Application | Dilution Range |
|---|---|
| Western Blot (WB) | 1:500-1:1000 |
| Immunohistochemistry (IHC) | 1:1000-1:4000 |
These dilution parameters should be considered starting points, as the manufacturer explicitly notes that optimal dilution is sample-dependent . Methodologically sound experimental design requires titration of the antibody in each specific testing system to determine optimal signal-to-noise ratios. For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative in some experimental contexts .
What are the optimal storage conditions for maintaining SP3 antibody stability?
SP3 antibody (26584-1-AP) exhibits greatest stability when stored at -20°C in its formulation buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibody maintains functionality for one year post-shipment. Unlike many research antibodies, aliquoting is unnecessary for -20°C storage, which significantly simplifies laboratory reagent management. For the 20μl size preparations, the product contains 0.1% BSA as an additional stabilizing agent .
How does SP3 antibody for HER2 detection compare with other established antibodies?
Comparative evaluation of SP3 versus CB11 antibody for HER2 detection demonstrated a statistically significant correlation (p<0.001) with an 86.9% concordance rate in a study of 190 breast carcinoma cases . The staining characteristics between these antibodies revealed important methodological differences:
| SP3 Antibody Characteristics | CB11 Antibody Characteristics |
|---|---|
| Strong complete membrane staining | Frequent non-specific cytoplasmic staining |
| Reduced background interference | Higher background signal |
| Lower interobserver variability | Greater interobserver variability |
When cases scored as negative/1+ were grouped and 2+ cases excluded, correlation remained strong with only four discordant cases identified . In a separate evaluation comparing SP3 with HercepTest, after excluding equivocal (2+) cases, concordance reached 97.6% (95% CI 94.0% to 99.3%) with κ=0.88 (95% CI 0.77 to 1.00) .
What is the concordance between SP3 antibody results and gene amplification methodologies?
Research data demonstrates robust concordance between SP3 immunohistochemistry and molecular techniques:
With CISH (Chromogenic In Situ Hybridization) as reference:
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23/24 (95.8%) of SP3 3+ cases showed gene amplification
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97.3% of cases without gene amplification were SP3 negative
With FISH (Fluorescence In Situ Hybridization) as reference:
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Concordance in needle core biopsy specimens: 96% (95% CI 91.9% to 99.7%) with κ=0.89
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Concordance in excisional biopsy specimens: 97% (95% CI 90.3% to 99.3%) with κ=0.84
These high concordance metrics establish SP3 as a reliable indicator of HER2 gene amplification status, validating its utility in research and diagnostic applications.
What factors influence SP3 antibody specificity and sensitivity?
SP3 antibody for HER2 detection demonstrates exceptional specificity (99.1% with CISH as standard ; 100% with FISH as reference ) but moderate sensitivity (52.3% with CISH ; 78.3% with FISH ). This performance profile is influenced by several experimental and biological variables:
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Epitope accessibility: The potential cleavage of HER2 with loss of the extracellular domain explains some false negative cases observed with SP3 . Since SP3 targets the extracellular domain, shedding of the binding site might lead to negative/1+ IHC classification despite gene amplification.
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Tissue fixation parameters: Fixative type, duration, and pH significantly impact epitope preservation and antibody binding efficiency.
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Antigen retrieval methodology: Optimization with TE buffer pH 9.0 or alternative citrate buffer pH 6.0 can significantly affect staining intensity and specificity .
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Sample preparation variables: An 89% concordance rate between SP3 results in needle core biopsies versus excisional biopsies suggests sample preparation influences results .
The complete performance characteristics are summarized in the following table:
| Parameter | SP3 | CB11 |
|---|---|---|
| Sensitivity | 0.52 | 0.52 |
| Specificity | 0.99 | 0.98 |
| Positive Predictive Value | 0.96 | 0.92 |
| Negative Predictive Value | 0.84 | 0.93 |
What are the advantages of rabbit monoclonal antibodies like SP3 compared to mouse monoclonals?
SP3 belongs to a class of immunoreagents that combine advantageous properties of both mouse monoclonal antibodies and rabbit antisera . The specific advantages include:
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Superior affinity and sensitivity: Rabbit-derived antibodies typically demonstrate 10-100 fold higher antigen affinity compared to mouse antibodies.
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Enhanced specificity: Reduced cross-reactivity while maintaining high signal strength.
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Recognition of epitopes that may be non-immunogenic in mice due to phylogenetic differences.
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Improved signal-to-noise ratio: SP3 produces cleaner membrane staining with minimal cytoplasmic background compared to mouse monoclonal antibodies like CB11 .
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Better tissue penetration properties: Molecular characteristics facilitate improved penetration into formalin-fixed, paraffin-embedded tissues.
These properties make SP3 particularly valuable for detecting limited epitope presentation or in challenging tissue environments.
How can researchers optimize SP3 antibody protocols for different experimental systems?
Protocol optimization for SP3 antibody across diverse experimental systems requires systematic adjustment of multiple parameters:
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Antigen retrieval optimization: For IHC applications, comparative testing between TE buffer pH 9.0 and citrate buffer pH 6.0 to determine optimal epitope exposure for specific tissue types .
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Titration analysis: While recommended dilutions provide starting points (1:500-1:1000 for WB; 1:1000-1:4000 for IHC), systematic titration series should be conducted to determine optimal concentration for each experimental system .
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Incubation parameter modification: Adjusting time and temperature variables can enhance sensitivity while maintaining specificity in challenging tissues.
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Detection system selection: Alternative detection methodologies may be necessary for tissues with high endogenous peroxidase or biotin levels.
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Tissue-specific blocking optimization: Modification of blocking reagents can reduce background in high-autofluorescence tissues.
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Cross-platform validation: The observed 89% concordance between SP3 results in needle core biopsies versus excisional biopsies highlights the importance of validating findings across different sample preparation methodologies .
What controls should be incorporated in SP3 antibody experimental protocols?
Rigorous experimental design with SP3 antibody requires inclusion of multiple control conditions:
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Positive control samples: For SP3 transcription factor antibody, PC-3 cells, C2C12 cells, or COLO 320 cells for Western blotting; mouse lung tissue or human ovary cancer tissue for IHC .
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Negative control tissues: Samples with confirmed absence of target protein expression.
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Isotype control: Non-specific rabbit IgG at equivalent concentration as SP3 antibody to assess non-specific binding.
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Absorption control: Pre-incubation of antibody with target antigen should abolish specific staining.
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Secondary antibody-only control: Omission of primary antibody to detect non-specific secondary antibody binding.
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Loading controls for Western blotting: Housekeeping protein detection to ensure equal protein loading.
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Reference standards: For HER2 detection, inclusion of cases with established HER2 status determined by FISH or CISH.
Implementation of this comprehensive control strategy enables distinction between specific and non-specific signals, validating experimental findings.
What molecular weight patterns should researchers expect when working with SP3 antibody?
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Presence of multiple isoforms
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Post-translational modifications
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Proteolytic processing
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Protein-protein interactions altering electrophoretic mobility
When analyzing Western blot results, researchers should anticipate these molecular weight patterns and investigate aberrant banding patterns to determine whether they represent specific signal or experimental artifacts.
How do post-translational modifications affect SP3 detection and function?
The SP3 transcription factor undergoes several post-translational modifications that significantly impact antibody detection and protein function:
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SUMOylation: SP3 is sumoylated on all isoforms, with certain isoforms containing 2 distinct SUMOylation sites . This modification influences SP3 subcellular localization, directing it to the nuclear periphery and specific nuclear dots, including some localization in PML nuclear bodies .
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Acetylation: SP3 is dynamically regulated through acetylation by histone acetyltransferase p300 and deacetylation by HDACs . These acetylation states may alter protein conformation and epitope accessibility.
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Additional modifications: Phosphorylation and other modifications likely contribute to the observed molecular weight discrepancies between calculated (82 kDa) and experimentally observed (70 kDa, 115 kDa) masses .
These modifications explain the complex banding patterns observed in Western blotting and demonstrate why detection efficiency varies based on cellular context and functional state of the protein.
How reliable is SP3 antibody for HER2 status determination in breast cancer research?
Evidence strongly supports SP3 as a reliable alternative to HercepTest in evaluating HER2 status in breast cancer specimens. The data shows:
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High concordance (97.6%) between SP3 and HercepTest after excluding equivocal (2+) cases
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Strong agreement with gene amplification methods: 96-97% concordance with FISH in both needle core and excisional biopsies
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Excellent specificity (99-100%) for detecting HER2 overexpression/gene amplification
Research concludes that "SP3 is a reliable alternative to HercepTest in evaluating HER-2 status in breast cancer patients" and may serve as a valuable diagnostic tool in breast pathology with potential utility as an IHC biomarker in non-mammary malignancies as well.
What are the potential sources of discrepancy between SP3 immunohistochemistry and gene amplification methods?
Several biological and technical factors may explain discordances between SP3 antibody staining and molecular HER2 testing:
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Extracellular domain shedding: Since SP3 targets the HER2 extracellular domain, proteolytic processing of this region results in negative staining despite gene amplification . This biological mechanism explains the moderate sensitivity (52.3-78.3%) contrasted with high specificity (99-100%) .
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Tumor heterogeneity: Spatial variation in HER2 expression within tumors leads to sampling discrepancies between tissues used for IHC versus those analyzed by FISH/CISH.
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Post-translational modifications affecting epitope accessibility.
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Methodological variables: Fixation protocols, processing parameters, and staining techniques introduce technical variability.
Understanding these mechanisms is critical for accurate result interpretation in research and diagnostic applications.
