SERBP1 Antibody

What is SERBP1 and what cellular functions does it perform?

SERBP1 (SERPINE1 mRNA-binding protein 1), also known as PAI-RBP1, CGI-55, CHD3IP, or HABP4L, is a membrane-associated protein with multiple cellular functions. Its primary role involves regulating mRNA stability by specifically binding to the cyclic nucleotide-responsive sequence (CRS) motif of PAI-1 mRNA, stabilizing this transcript and regulating its expression . SERBP1 contains arginine-glycine (RG)-rich and arginine-glycine-glycine (RGG) box domains for target mRNA binding, though it lacks typical RNA recognition motif (RRM) or K homology (KH) domains . Beyond RNA binding, SERBP1 interacts with Mi2-alpha and may participate in chromatin remodeling processes . It also interacts with PGRMC1 and mediates progesterone's antiapoptotic effects in ovarian cell types . Recent research has demonstrated SERBP1's involvement in homologous recombination-mediated DNA repair in response to double-strand breaks through regulation of CtIP translation during S phase .

Where does SERBP1 localize within cells?

SERBP1 exhibits a complex subcellular distribution pattern across multiple compartments. It localizes primarily to the nucleus, perinuclear region of the cytoplasm, and plasma membrane . This multi-compartment distribution reflects its diverse cellular functions. Interestingly, while SERBP1 predominantly resides in the cytoplasm, it interacts with numerous nuclear proteins including CHD3, Daxx, Topors, and PIASy . This interaction profile suggests SERBP1 may shuttle between compartments or participate in nucleocytoplasmic transport processes. The specific localization pattern may vary depending on cell type, cell cycle phase, and physiological conditions, highlighting the need for careful immunolocalization studies when investigating SERBP1 functions.

What applications are SERBP1 antibodies validated for?

SERBP1 antibodies have been validated for multiple research applications with specific recommendations for optimal results:

For immunohistochemistry applications using formalin-fixed paraffin-embedded tissues, antigen retrieval is critical. The recommended protocol involves heating tissue sections in 10mM Tris with 1mM EDTA (pH 9.0) for 45 minutes at 95°C, followed by cooling at room temperature for 20 minutes . Western blot analysis has been successfully performed on cell lines including K562 and PC3, which can serve as positive controls . Most commercially available SERBP1 antibodies are mouse monoclonals with IgG2b or IgG2c kappa isotypes .

How does SERBP1 influence homologous recombination-mediated DNA repair?

SERBP1 plays a significant role in homologous recombination (HR)-mediated DNA repair, specifically in response to DNA double-strand breaks (DSBs). The mechanistic basis of this function involves regulation of CtIP translation during S phase of the cell cycle . CtIP is a critical component of the DNA end resection machinery, which is an essential initial step in HR-mediated repair. SERBP1, functioning as an RNA-binding protein, regulates the translation efficiency of CtIP mRNA, thereby controlling CtIP protein levels available for DNA repair processes . This regulatory activity connects SERBP1's RNA-binding capabilities directly to genome stability maintenance. The temporal regulation during S phase is particularly significant, as this is when sister chromatids are available as templates for homologous recombination. This discovery highlights SERBP1 as an important factor linking post-transcriptional gene regulation to DNA damage response pathways.

What is known about SERBP1's role in cancer progression?

SERBP1 has significant implications in cancer biology, with evidence pointing to its involvement in both tumorigenesis and therapy resistance. Overexpression of SERBP1 has been documented across multiple cancer types including:

Beyond its diagnostic and prognostic implications, SERBP1 has been linked to therapeutic resistance. In several human cancer cell lines, increased SERBP1 expression has been observed in cisplatin-resistant variants compared to their cisplatin-sensitive counterparts . This suggests SERBP1 may contribute to chemoresistance mechanisms, possibly through its roles in mRNA stability regulation, chromatin remodeling, or DNA repair. The association with DNA repair pathways is particularly relevant, as enhanced DNA repair capacity is a known mechanism of resistance to DNA-damaging chemotherapeutics.

What protein-protein interactions are important for SERBP1 function?

SERBP1 engages in numerous protein-protein interactions that expand our understanding of its cellular functions. Key interaction partners have been identified using yeast two-hybrid screening and other protein interaction detection methods:

The interaction with nuclear proteins is particularly intriguing given SERBP1's predominantly cytoplasmic localization . This suggests SERBP1 may shuttle between compartments or participate in nucleocytoplasmic transport processes. These diverse interactions position SERBP1 at the intersection of multiple cellular pathways including chromatin remodeling, transcriptional regulation, hormone signaling, and post-translational modification processes. Investigating how these interactions are regulated in different cellular contexts remains an important area for future research.

How should researchers troubleshoot non-specific binding with SERBP1 antibodies?

When encountering non-specific binding issues with SERBP1 antibodies, researchers should implement a systematic troubleshooting approach:

• Antibody validation strategies:

  • Use multiple antibodies targeting different SERBP1 epitopes to confirm specificity

  • Include positive controls (cell lines with known SERBP1 expression such as K562 or PC3)

  • Incorporate negative controls (SERBP1 knockdown samples)

  • Perform peptide competition assays to confirm specificity

• Protocol optimization:

  • Titrate antibody concentration (starting with recommended 1-2 μg/ml and adjusting as needed)

  • Modify blocking conditions (test different blocking agents: BSA, non-fat milk, normal serum)

  • Increase washing stringency (longer washes, higher detergent concentration)

  • For Western blotting: verify molecular weight (expected ~60 kDa)

  • For IHC: optimize antigen retrieval conditions (10mM Tris with 1mM EDTA, pH 9.0)

• Sample-specific considerations:

  • Ensure proper sample preparation (appropriate lysis buffers, protease inhibitors)

  • For tissues, assess fixation quality and optimize processing steps

  • Consider tissue-specific autofluorescence or endogenous peroxidase activity

  • Test fresh vs. stored antibody aliquots to rule out degradation issues

What approaches can determine if SERBP1 directly regulates a specific mRNA target?

To establish direct regulation of a specific mRNA by SERBP1, researchers should employ a comprehensive experimental strategy:

• RNA-binding assessment:

  • RNA immunoprecipitation (RIP) using SERBP1 antibodies to isolate bound mRNAs

  • CLIP-seq (Crosslinking immunoprecipitation-sequencing) to identify direct binding sites

  • In vitro binding assays with recombinant SERBP1 and candidate mRNA sequences

  • Focus on CRS (cyclic nucleotide-responsive sequence) motifs as known SERBP1 binding sites

• Functional validation:

  • mRNA stability assays following SERBP1 knockdown or overexpression

  • Actinomycin D chase experiments to measure mRNA half-life changes

  • Reporter assays with wild-type and mutated SERBP1 binding sites

  • Polysome profiling to assess translation efficiency changes

• Structural analysis:

  • Map the specific binding region in both SERBP1 protein (focusing on RG-rich and RGG box domains)

  • Identify the precise RNA sequence elements required for interaction

  • Determine if binding is direct or mediated through protein complexes

What is the optimal protocol for detecting SERBP1 by Western blot?

For reliable SERBP1 detection by Western blot, the following optimized protocol is recommended:

• Sample preparation:

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Quantify protein concentration (BCA or Bradford assay)

  • Prepare 20-40 μg total protein per lane in Laemmli buffer with reducing agent

  • Heat samples at 95°C for 5 minutes

• Gel electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE (60 kDa target)

  • Transfer to PVDF or nitrocellulose membrane (0.45 μm pore size)

  • Verify transfer efficiency with Ponceau S staining

• Immunoblotting:

  • Block membrane with 5% non-fat milk or BSA in TBST (1 hour, room temperature)

  • Incubate with SERBP1 primary antibody at 1-2 μg/ml dilution overnight at 4°C

  • Wash 3× with TBST (10 minutes each)

  • Incubate with HRP-conjugated anti-mouse secondary antibody (1:5000, 1 hour, room temperature)

  • Wash 3× with TBST (10 minutes each)

  • Develop using ECL substrate and image

• Controls and validation:

  • Include positive control (K562 or PC3 cell lysates)

  • Run molecular weight markers to confirm 60 kDa band

  • Consider siRNA knockdown controls for antibody validation

  • Include loading control (β-actin, GAPDH, or total protein stain)

What is the recommended procedure for SERBP1 immunohistochemistry?

For optimal SERBP1 detection in formalin-fixed paraffin-embedded (FFPE) tissues:

• Tissue preparation and sectioning:

  • Fix tissues in 10% neutral buffered formalin (24-48 hours)

  • Process and embed in paraffin using standard histology protocols

  • Section tissues at 4-5 μm thickness onto adhesive slides

  • Air-dry sections overnight at room temperature

• Deparaffinization and antigen retrieval (critical step):

  • Deparaffinize in xylene and rehydrate through graded alcohols

  • Perform heat-induced epitope retrieval in 10mM Tris with 1mM EDTA, pH 9.0

  • Heat for 45 minutes at 95°C followed by 20 minutes cooling at room temperature

  • Wash thoroughly in buffer

• Immunostaining procedure:

  • Block endogenous peroxidase (3% H₂O₂, 10 minutes)

  • Apply protein block (10-30 minutes)

  • Incubate with SERBP1 antibody at 1-2 μg/ml for 30 minutes at room temperature

  • Wash 3× in buffer

  • Apply detection system (polymer-HRP recommended)

  • Develop with DAB chromogen

  • Counterstain with hematoxylin, dehydrate and mount

• Controls and validation:

  • Include known positive tissue (kidney shows positive SERBP1 staining)

  • Include isotype control (mouse IgG2b or IgG2c depending on clone)

  • Consider subcellular localization (nuclear, perinuclear cytoplasm, and membrane)

How should researchers design experiments to study SERBP1's role in DNA repair?

To effectively investigate SERBP1's function in DNA repair processes, researchers should design experiments that address multiple aspects of this relationship:

• Expression manipulation studies:

  • Generate SERBP1 knockdown cell lines (siRNA, shRNA, or CRISPR-Cas9)

  • Create SERBP1 overexpression models (wild-type and functional domain mutants)

  • Develop cell cycle-specific expression systems (S-phase specific)

  • Include rescue experiments with RNAi-resistant constructs

• DNA damage and repair assays:

  • Induction methods: ionizing radiation, etoposide, or site-specific nucleases

  • HR efficiency measurement using DR-GFP reporter system

  • RAD51 foci formation quantification by immunofluorescence

  • Comet assay to assess DNA break resolution kinetics

  • CtIP protein levels by Western blot (SERBP1 regulates CtIP translation)

• Mechanistic investigations:

  • RNA immunoprecipitation to assess SERBP1 binding to CtIP mRNA

  • Polysome profiling to evaluate translation efficiency of CtIP

  • Time-course experiments following synchronized cell populations

  • Co-immunoprecipitation to identify DNA repair protein interactions

• Validation in multiple models:

  • Different cell types (cancer vs. normal cells)

  • Primary vs. established cell lines

  • Correlation with clinical samples (tumors with different SERBP1 expression levels)

What controls are essential when studying SERBP1 in cancer models?

When investigating SERBP1 in cancer contexts, incorporating comprehensive controls is crucial:

• Expression controls:

  • Matched normal vs. tumor tissue from the same patient

  • Panel of cancer cell lines with varied SERBP1 expression levels

  • Non-transformed cell lines as baseline controls

  • Positive control tissues (kidney shows reliable SERBP1 staining)

• Technical controls:

  • Multiple SERBP1 antibodies targeting different epitopes

  • Isotype-matched negative controls for immunostaining

  • SERBP1 knockdown samples as specificity controls

  • Blocking peptide competition for antibody validation

• Functional validation controls:

  • Rescue experiments with wild-type vs. mutant SERBP1

  • Dose-dependent response assessment

  • Time-course experiments to capture dynamic processes

  • Correlate with established cancer biomarkers

• Cancer-specific considerations:

  • Stratify by cancer type, grade, and stage (SERBP1 is associated with high-grade ovarian tumors)

  • Consider microenvironmental factors

  • Assess in treatment-naïve vs. treatment-resistant models (SERBP1 is associated with cisplatin resistance)

  • Evaluate in metastatic vs. primary tumor samples (SERBP1 correlates with metastatic potential in lung cancer)

How can SERBP1's influence on mRNA stability be quantitatively measured?

To quantitatively assess SERBP1's impact on mRNA stability:

• mRNA decay kinetics measurement:

  • Actinomycin D chase experiments in SERBP1-manipulated cells

  • Collect RNA samples at regular intervals (0, 1, 2, 4, 8, 12 hours)

  • Quantify target mRNA levels by RT-qPCR

  • Calculate half-life using exponential decay modeling

  • Focus on known targets (PAI-1 mRNA contains CRS motif)

• Direct binding assessment:

  • RNA immunoprecipitation with SERBP1 antibodies

  • qPCR quantification of co-precipitated mRNAs

  • RNase protection assays to map binding sites

  • In vitro binding assays with recombinant SERBP1 and synthetic RNA

• Reporter systems:

  • Luciferase constructs with wild-type and mutated SERBP1 binding sites

  • Pulse-chase labeling of nascent RNA

  • MS2-GFP system for real-time visualization of mRNA decay

  • CRISPR-Cas13 RNA labeling for live cell imaging

• Comprehensive transcriptomic analysis:

  • RNA-seq in SERBP1 knockdown vs. control cells

  • Focus on differential expression of mRNAs containing CRS motifs

  • Integration with CLIP-seq data to identify direct targets

  • Pathway analysis to identify biological processes affected

How should variable SERBP1 expression patterns across tissues be interpreted?

When analyzing differential SERBP1 expression across tissues, consider these interpretive frameworks:

• Analytical approach:

  • Normalize expression data consistently (protein loading, housekeeping genes)

  • Apply appropriate statistical tests for multi-tissue comparisons

  • Consider both staining intensity and percentage of positive cells

  • Distinguish between nuclear, cytoplasmic, and membrane localization

• Physiological context:

  • Correlate with tissue-specific functions (proliferation rate, differentiation status)

  • Consider relationship to RNA metabolism in different tissues

  • Evaluate co-expression patterns with interacting partners (CHD3, PGRMC1)

  • Assess relationship to hormone responsiveness (SERBP1 mediates progesterone effects)

• Pathological implications:

  • Compare SERBP1 expression in normal vs. disease states

  • Analyze correlation with cancer progression (SERBP1 overexpression in various cancers)

  • Stratify by clinical parameters in cancer tissues (grade, stage, metastatic status)

  • Evaluate potential as a biomarker for specific pathologies

• Mechanistic considerations:

  • Assess correlation with DNA repair efficiency in different tissues

  • Examine relationship to mRNA stability of tissue-specific transcripts

  • Consider regulation by tissue-specific transcription factors

  • Evaluate potential feedback mechanisms affecting SERBP1 levels

What explains variation in SERBP1 protein detection by different techniques?

Variations in SERBP1 detection across different experimental methods may result from:

• Technical factors:

  • Antibody epitope accessibility varies between applications

  • Fixation and processing effects on antigen preservation (particularly for IHC)

  • Denaturation state differences (WB vs. immunofluorescence)

  • Detection system sensitivity thresholds

• Biological considerations:

  • Post-translational modifications affecting epitope recognition

  • Alternative splicing generating different isoforms

  • Complex formation masking antibody binding sites

  • Subcellular compartmentalization (nuclear, perinuclear, and membrane localization)

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Apply complementary techniques (WB, IHC, IF, flow cytometry)

  • Include appropriate positive and negative controls for each method

  • Validate findings with genetic approaches (knockdown/overexpression)

  • Consider cell-type specific factors affecting detection

• Reporting recommendations:

  • Clearly document detection method parameters

  • Specify antibody clone, dilution, and incubation conditions

  • Include complete methodology for sample preparation

  • Present raw data alongside processed/quantified results

How should researchers analyze SERBP1 immunostaining in tumor tissues?

For comprehensive analysis of SERBP1 immunohistochemistry in tumor specimens:

• Quantitative assessment:

  • Apply standardized scoring systems (H-score, Allred score)

  • Calculate percentage of positive cells in representative fields

  • Grade staining intensity (0-3+ scale)

  • Consider digital image analysis for objective quantification

• Pattern analysis:

  • Evaluate subcellular localization (nuclear, cytoplasmic, membrane)

  • Assess distribution pattern (homogeneous vs. heterogeneous)

  • Document relationship to histological features (tumor borders, necrotic areas)

  • Compare with normal adjacent tissue when available

• Clinical correlation:

  • Stratify by tumor type, grade, and stage (SERBP1 associated with high-grade ovarian tumors)

  • Correlate with patient outcome data (survival, recurrence)

  • Analyze relationship to treatment response (SERBP1 linked to cisplatin resistance)

  • Assess association with metastatic status (SERBP1 correlates with metastatic potential)

• Multimarker approaches:

  • Co-stain with proliferation markers (Ki67)

  • Analyze relationship to DNA damage response proteins (γH2AX, RAD51)

  • Correlate with known SERBP1 interactors (CHD3, PGRMC1)

  • Incorporate into broader biomarker panels for enhanced prognostic value

What statistical approaches are appropriate for SERBP1 expression studies?

When analyzing SERBP1 expression data, consider these statistical approaches:

• Descriptive statistics:

  • Mean, median, and range of expression values

  • Standard deviation or interquartile range for dispersion

  • Frequency distributions to identify expression patterns

  • Box plots stratified by relevant categories (tissue type, cancer grade)

• Comparative analyses:

  • t-tests for two-group comparisons (normal vs. tumor)

  • ANOVA with post-hoc tests for multi-group comparisons

  • Non-parametric alternatives when normality cannot be assumed

  • Paired tests for matched sample comparisons

• Correlation studies:

  • Pearson correlation for normally distributed continuous variables

  • Spearman rank correlation for non-parametric data

  • Partial correlation to control for confounding variables

  • Multiple regression to identify independent predictors of SERBP1 expression

• Survival analysis:

  • Kaplan-Meier curves stratified by SERBP1 expression levels

  • Log-rank tests for survival curve comparison

  • Cox proportional hazards models for multivariate analysis

  • Time-dependent ROC analysis for prognostic performance

• Statistical rigor considerations:

  • A priori sample size and power calculations

  • Multiple testing correction (Benjamini-Hochberg method)

  • Report effect sizes alongside p-values

  • Validation in independent cohorts when possible

How can researchers integrate SERBP1 findings with broader omics datasets?

To maximize the value of SERBP1 research within the context of larger omics investigations:

• Multi-omics integration approaches:

  • Correlate SERBP1 protein expression with transcriptomic data

  • Analyze relationship to proteome-wide changes in SERBP1-manipulated models

  • Incorporate phosphoproteomic data to assess signaling pathway effects

  • Evaluate impact on metabolomic profiles

• Pathway analysis:

  • Gene set enrichment analysis to identify affected biological processes

  • Network analysis to position SERBP1 within functional interaction webs

  • Focus on RNA metabolism and DNA repair pathways (known SERBP1 functions)

  • Identify potential feedback mechanisms regulating SERBP1 levels

• Public database utilization:

  • Mine TCGA and GTEx datasets for SERBP1 expression patterns

  • Compare with protein expression data from Human Protein Atlas

  • Analyze correlation with clinical parameters in patient cohorts

  • Identify potential therapeutic targets within SERBP1-associated networks

• Systems biology perspectives:

  • Develop predictive models of SERBP1's role in cellular homeostasis

  • Simulate effects of SERBP1 perturbation on key cellular processes

  • Identify potential synthetic lethal interactions for therapeutic targeting

  • Construct temporal models of SERBP1 function across cell cycle phases