PSPC1 Antibody, Biotin conjugated

What is PSPC1 and what are its key biological functions?

PSPC1 is a paraspeckle biomarker and putative transcription factor belonging to the Drosophila behavior/human splicing (DBHS) family. Recent research has identified PSPC1 as a contextual determinant of tumor progression across multiple cancer types, involving oncogenic reprogramming that switches proapoptotic TGF-β to prometastatic TGF-β via hijacking of Smad2/3 targeting . The protein contains a multifunctional DBHS domain essential for its interactions with other nuclear factors, including TET1, as demonstrated through domain-mapping experiments .

Methodologically, researchers investigating PSPC1 should consider its dual DNA and RNA binding capabilities. PSPC1 has been shown to function in a molecular axis with TET1 and the lncRNA Neat1 to modulate PRC2-binding affinity to chromatin and bivalent gene transcripts in embryonic stem cells, contributing to stem cell bivalency . Additionally, PSPC1 has been identified as aberrantly overexpressed in AML patients, where it exerts pro-leukemic effects by regulating leukemic transcription programs through cooperative binding with PU.1 .

How do application-specific dilutions differ for biotin-conjugated PSPC1 antibodies compared to unconjugated versions?

While standard PSPC1 antibody applications follow established dilution protocols, biotin conjugation necessitates optimization for each experimental system. For unconjugated PSPC1 antibody (16714-1-AP), the recommended dilutions are:

For biotin-conjugated versions, researchers should perform titration experiments starting at similar dilutions but anticipate potential adjustments. Biotin conjugation can affect antibody binding kinetics and may require modified dilutions to maintain signal-to-noise ratios. The higher sensitivity of streptavidin-based detection systems typically allows for higher dilutions of biotin-conjugated antibodies, but this must be empirically determined for each experimental system.

What validated cell models exist for PSPC1 antibody applications?

Validation experiments have confirmed specific reactivity across multiple cell lines and applications for PSPC1 antibody:

For biotin-conjugated PSPC1 antibody applications, researchers should first validate performance in these established cell models before expanding to other systems. Additionally, PSPC1 antibodies have demonstrated reactivity with human, mouse, and rat samples, offering cross-species application potential . When studying PSPC1 in AML contexts, researchers can reference recent work demonstrating its aberrant overexpression in AML patient samples, which provides a pathological model system for antibody validation .

What storage and handling protocols maximize biotin-conjugated PSPC1 antibody performance?

Optimal storage conditions for PSPC1 antibodies include maintaining at -20°C in a buffer containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3). Under these conditions, the antibody remains stable for one year after shipment without requiring aliquoting . For biotin-conjugated versions, additional precautions are necessary:

• Avoid repeated freeze-thaw cycles which can compromise both antibody binding capacity and biotin-streptavidin interaction

• Store in amber tubes to protect biotin from light exposure

• Use BSA-containing buffers (0.1% BSA) to prevent non-specific binding

• Incorporate sodium azide (0.02%) to prevent microbial contamination

• Confirm biotin conjugation stability through periodic streptavidin binding tests

When handling biotin-conjugated antibodies, also avoid buffers containing free biotin and ensure experimental protocols account for potential endogenous biotin interference, particularly in cells with high metabolic activity.

What positive and negative controls should be included when using PSPC1 antibodies in research protocols?

Rigorous experimental design requires appropriate controls to validate PSPC1 antibody specificity and performance:

Positive controls:

• HEK-293, HepG2, or PC-3 cell lysates for Western blot applications

• PC-3 cells for immunofluorescence applications

• Recombinant PSPC1 protein (observed at 66 kDa despite calculated 59 kDa size)

Negative controls:

• PSPC1 knockout cell lines, such as the validated Pspc1KO ESCs (clones C4 and C9) described in published research

• Primary antibody omission controls with detection system only

• Isotype controls to assess non-specific binding

• For biotin-conjugated antibodies specifically: streptavidin-only controls to assess endogenous biotin signal

Additionally, researchers should consider competitive blocking experiments with unconjugated antibody prior to biotin-conjugated antibody application to confirm binding specificity.

How can biotin-conjugated PSPC1 antibodies enhance chromatin immunoprecipitation studies examining the PSPC1-TET1-PRC2 regulatory axis?

Chromatin immunoprecipitation (ChIP) studies have revealed that PSPC1 co-occupies genomic regions with TET1 and PRC2 components, with 56.9% (1,322/2,324) of PSPC1 peaks co-occupied by TET1 and SUZ12 . Biotin-conjugated PSPC1 antibodies offer several methodological advantages for investigating these complex interactions:

• Enhanced ChIP-seq sensitivity: Biotin-streptavidin interactions provide stronger binding than standard antibody-protein A/G interactions, potentially increasing signal-to-noise ratios in ChIP experiments

• Sequential ChIP (ChIP-reChIP) applications: Biotin conjugation enables stringent elution using biotin competition rather than harsh denaturing conditions, preserving epitopes for subsequent immunoprecipitation with TET1 or PRC2 component antibodies

• Multiplex ChIP-seq approaches: The use of differently conjugated antibodies (biotin-PSPC1 with another tag for TET1) allows simultaneous immunoprecipitation to directly investigate co-occupancy

For optimal implementation, researchers should:

• Use formaldehyde crosslinking optimized for preserving both DNA-protein and protein-protein interactions

• Employ sonication conditions that generate 200-500bp fragments

• Include input controls and IgG controls for normalization

• Validate enrichment at known PSPC1 binding sites, particularly at bivalent gene promoters where 74.2% of PSPC1-binding peaks are located

What methodological strategies resolve contradictory findings between biochemical fractionation and immunofluorescence localization of PSPC1?

PSPC1 exhibits complex nuclear localization patterns, functioning both in paraspeckles and at chromatin, which can lead to seemingly contradictory results between different experimental approaches. To reconcile such discrepancies:

• Extraction method optimization: Biochemical fractionation results depend critically on extraction conditions. The DBHS domain of PSPC1 mediates protein-protein interactions that may resist standard extraction buffers . Sequential extraction with increasing detergent strengths can provide fractionation profiles that better align with microscopy observations.

• Fixation method comparison: For immunofluorescence, compare paraformaldehyde (preserves structure) with methanol (better penetration) fixation using biotin-conjugated PSPC1 antibodies. Different fixation methods may preferentially preserve distinct PSPC1 subpopulations.

• Proximity-based mapping: Employ BioID fusion with PSPC1 for proximity labeling of interacting proteins in living cells, providing an orthogonal approach to determine PSPC1 associations that is independent of extraction or fixation artifacts.

• Sequential imaging-biochemistry workflow: Perform live-cell imaging of fluorescently-tagged PSPC1 followed by fractionation of the same cell population, establishing direct correspondence between visualization and biochemical data.

• Super-resolution microscopy: Conventional microscopy may not resolve PSPC1 distribution patterns adequately. Techniques like STORM or STED combined with biotin-conjugated PSPC1 antibodies can provide nanometer-scale localization data that better aligns with biochemical findings.

How can researchers apply PSPC1 antibodies in studying its role in acute myeloid leukemia (AML) progression and potential therapeutic targeting?

Recent research has identified PSPC1 as aberrantly overexpressed in AML patients and associated with poor survival outcomes. PSPC1 has been shown to be critical for maintaining leukemic characteristics, with its loss inducing differentiation, suppressing proliferation, and abolishing leukemogenesis . Methodological approaches using PSPC1 antibodies to investigate this role include:

• Patient stratification studies: Using immunohistochemistry with biotin-conjugated PSPC1 antibodies on AML patient samples to correlate expression levels with clinical outcomes, disease subtypes, and treatment responses.

• Multi-parameter flow cytometry: Develop protocols combining biotin-conjugated PSPC1 antibodies with lineage markers to quantify PSPC1 expression across different AML blast populations and monitor changes during differentiation therapy.

• ChIP-seq profiling of PSPC1-PU.1 cooperation: Since PSPC1 exerts pro-leukemia effects through cooperative chromatin binding with PU.1 , ChIP-seq using biotin-conjugated PSPC1 antibodies can map this cooperative binding genome-wide in AML cells.

• Target gene validation: Confirm PSPC1 binding to critical target genes like NDC1 (identified as a PSPC1-regulated gene in AML not previously implicated in this disease) using ChIP-qPCR with biotin-conjugated PSPC1 antibodies.

• Therapy response monitoring: Develop immunoassays using biotin-conjugated PSPC1 antibodies to measure PSPC1 levels in patient samples during treatment, potentially serving as a biomarker for therapeutic response.

What approaches can effectively validate PSPC1 antibody specificity in knockout/knockdown experimental systems?

Rigorous antibody validation is essential for reliable experimental outcomes. For PSPC1 antibodies, comprehensive validation should include:

• Western blot analysis with genetic controls: Compare antibody reactivity between wildtype and PSPC1 knockout/knockdown samples. Expect detection of a 66 kDa band (the observed molecular weight despite calculated 59 kDa) in wildtype samples that is reduced or absent in knockout/knockdown samples.

• Immunoprecipitation-mass spectrometry validation: Perform IP with PSPC1 antibodies followed by mass spectrometry of the immunoprecipitated proteins from both wildtype and knockout cells. This approach can identify both specific targets and potential cross-reactive proteins.

• ChIP-qPCR at known binding sites: Validate antibody specificity through ChIP-qPCR at established PSPC1 binding regions using wildtype and knockout cells. The research indicates PSPC1 binds primarily at gene promoters (74.2% of binding peaks) , providing specific regions for validation.

• Immunofluorescence pattern analysis: Compare nuclear paraspeckle staining patterns between wildtype and knockout cells. For biotin-conjugated antibodies specifically, include additional controls with free biotin pre-blocking to confirm signal specificity.

• Multiple antibody comparison: Use multiple PSPC1 antibodies targeting different epitopes and compare their binding profiles in both presence and absence of PSPC1 knockout/knockdown.

What protocols optimize the use of biotin-conjugated PSPC1 antibodies for multiplex imaging with other paraspeckle components?

Paraspeckles contain multiple protein and RNA components that function in coordinated complexes. Optimized multiplex imaging with biotin-conjugated PSPC1 antibodies requires:

• Sequential staining protocol:

  • First round: Incubate with biotin-conjugated PSPC1 antibody (1:100-1:200 dilution)

  • Detect with streptavidin conjugated to a far-red fluorophore (e.g., Alexa Fluor 647)

  • Perform stringent washing with high-salt buffer

  • Second round: Incubate with antibodies against other paraspeckle proteins (NONO, SFPQ)

  • Detect with spectrally compatible fluorophores

• RNA-protein co-detection:

  • For NEAT1 lncRNA detection (the essential structural component of paraspeckles mentioned in research ), perform RNA-FISH first

  • Follow with biotin-conjugated PSPC1 antibody immunofluorescence

  • Include RNase controls to confirm RNA-dependent structures

• Proximity ligation assay (PLA) optimization:

  • Use biotin-conjugated PSPC1 antibody with unconjugated antibodies against interaction partners (TET1, PRC2 components)

  • Detect with streptavidin-oligonucleotide conjugates and standard PLA protocols

  • This approach visualizes specific PSPC1 protein-protein interactions in situ with high sensitivity

• 3D imaging considerations:

  • Acquire z-stacks (0.2-0.3 μm steps) to capture entire nuclear volume

  • Perform deconvolution to enhance signal-to-noise ratio

  • Analyze paraspeckle number, size, and co-localization coefficients in 3D reconstructions

This multiplex approach permits investigation of the complex relationship between PSPC1 and other factors in the TET1-PSPC1-Neat1 molecular axis described in the research , providing spatial context to biochemical interaction data.

What factors influence the observed molecular weight discrepancy between calculated and experimental values for PSPC1?

The observed molecular weight of PSPC1 in Western blot applications (66 kDa) differs from its calculated value (59 kDa based on 523 amino acids) . This discrepancy requires methodological consideration:

• Post-translational modifications: PSPC1 undergoes modifications including phosphorylation, methylation, and SUMOylation that increase its apparent molecular weight. When troubleshooting antibody specificity issues, researchers should:

  • Include phosphatase-treated controls to assess contribution of phosphorylation

  • Consider sample preparation methods that preserve or remove specific modifications

  • Compare migration patterns across different cell types and conditions

• Isoform detection: Multiple PSPC1 isoforms exist due to alternative splicing. Biotin-conjugated antibodies may detect specific isoforms depending on epitope location. Researchers should:

  • Consult epitope mapping data to determine which isoforms will be recognized

  • Run high-resolution gels to separate closely migrating isoforms

  • Consider isoform-specific primers for RT-PCR validation alongside antibody detection

• Sample preparation influence: Extraction methods and buffer compositions affect observed migration patterns. Optimize by:

  • Comparing different lysis buffers (RIPA vs. NP-40 vs. urea-based)

  • Testing various reducing agent concentrations

  • Assessing the impact of heat denaturation time and temperature

• Gel system considerations: Polyacrylamide percentage and buffer systems influence migration. Test:

  • Different acrylamide percentages (8-12%) for optimal resolution

  • Gradient gels to better separate proteins in the 50-70 kDa range

  • Both Tris-glycine and Tris-tricine buffer systems for optimal separation

How can researchers optimize biotin-conjugated PSPC1 antibody performance in chromatin studies investigating bivalent gene regulation?

PSPC1 has been shown to play important roles in regulating bivalent genes in stem cells through association with TET1 and PRC2 . For optimal chromatin studies with biotin-conjugated PSPC1 antibodies:

• Chromatin preparation optimization:

  • Crosslinking time: Test 5-15 minutes of formaldehyde fixation to preserve protein-DNA interactions without creating excessive crosslinks

  • Sonication conditions: Optimize sonication to produce 200-300bp fragments for high-resolution binding site identification

  • Chromatin quality assessment: Verify fragment size distribution by gel electrophoresis before immunoprecipitation

• Biotin-streptavidin capture enhancement:

  • Pre-clear chromatin with unconjugated streptavidin beads to reduce background

  • Use streptavidin beads with low non-specific binding characteristics

  • Include a biotin blocking step before elution to reduce background

• Controls for bivalent region analysis:

  • Include ChIP for histone marks H3K4me3 and H3K27me3 as positive controls for bivalent domains

  • Perform parallel ChIP for TET1 and SUZ12 to confirm co-occupancy

  • Include input normalization and IgG controls

• Data analysis considerations:

  • Focus analysis on promoter regions (within 5kb of TSS) where 74.2% of PSPC1 binding occurs

  • Compare PSPC1 binding patterns in pluripotent versus differentiated states

  • Correlate binding with expression changes of bivalent genes during differentiation

• Validation of functional interactions:

  • Complement ChIP data with interaction studies (Co-IP, PLA) using biotin-conjugated PSPC1 antibodies

  • Perform sequential ChIP to confirm simultaneous binding of PSPC1 with TET1 or PRC2 components

  • Validate key findings in PSPC1 knockout models to establish causality