SRPK2 Antibody, Biotin conjugated

What is SRPK2 and why is it significant in neuroscience research?

SRPK2 (SRSF protein kinase 2) is a serine/threonine-protein kinase that specifically phosphorylates substrates rich in serine-arginine residues. This protein is widely expressed throughout the nervous system and has gained significant attention due to its upregulated expression in Alzheimer's Disease (AD) models. SRPK2 has been identified as a key modulating pathway in inflammatory processes related to neurodegeneration, making it an important target for investigating microglia-mediated neuroinflammation. Research has demonstrated that enhanced SRPK2 expression contributes directly to the proinflammatory activation of microglia, positioning it as a critical factor in AD pathology and potentially other neurodegenerative conditions .

What are the typical applications for SRPK2 Antibody, Biotin conjugated?

The SRPK2 Antibody, Biotin conjugated has been validated for several immunological applications, primarily ELISA (Enzyme-Linked Immunosorbent Assay), EIA (Enzyme Immunoassay), and other immunoassay techniques. The biotin conjugation provides significant advantages for detection sensitivity in these applications. For optimal results, researchers should use this antibody for human SRPK2 detection, as it has been specifically developed with human reactivity. When designing experiments, consider that this polyclonal antibody was raised against recombinant Human SRSF protein kinase 2 protein (specifically amino acids 471-688), which influences its epitope recognition properties .

How does SRPK2 Antibody perform in immunofluorescence studies of microglial cells?

In immunofluorescence applications for microglial research, SRPK2 antibodies can be effectively used to visualize SRPK2 expression patterns in relation to microglial activation markers. Published studies have successfully employed immunofluorescence techniques to demonstrate that SRPK2 expression correlates with increased levels of M1 polarization markers such as CD16/32 and IBA-1 in microglial cells. When conducting these experiments, researchers should include counterstaining with DAPI for nuclear visualization and incorporate quantitative analysis using image processing software like ImageJ to measure expression levels. For optimal results, cells should be fixed, properly blocked, and incubated with appropriate primary and secondary antibodies following standard immunofluorescence protocols .

How can SRPK2 Antibody be used to investigate the relationship between SRPK2 expression and microglial phenotype polarization?

To investigate the relationship between SRPK2 expression and microglial phenotype polarization, researchers should implement a comprehensive experimental design that manipulates SRPK2 levels while monitoring phenotype-specific markers. This approach requires:

• Manipulation of SRPK2 expression:

  • Overexpression using recombinant lentiviral vectors

  • Knockdown using targeted siRNA

• Assessment of phenotype markers:

  • M1 (proinflammatory) markers: CD16/32, IBA-1

  • M2 (anti-inflammatory) markers: CD206, Arg-1

• Morphological analysis:

  • Quantification of ameboid vs. ramified microglia

  • Cell size and process measurement

Research has demonstrated that SRPK2 enhancement significantly increases M1 marker expression while reducing M2 markers, corresponding with a higher percentage of ameboid microglia. Conversely, SRPK2 knockdown reduces M1 markers and promotes M2 phenotype characteristics. For quantification, ImageJ software can be employed to measure the percentage of cells expressing specific markers and to analyze morphological transformations .

What methodological approaches should be used to study the effect of SRPK2 on inflammatory cytokine production in microglia?

To thoroughly investigate SRPK2's impact on inflammatory cytokine production, a multi-modal approach combining gene expression analysis and protein quantification is recommended:

Experimental methodology:

• Genetic manipulation of SRPK2:

  • Enhanced expression through lentiviral vector transfection (SRPK2-en)

  • Reduced expression through siRNA transfection (SRPK2-KD)

• mRNA expression analysis:

  • Extract total RNA from treated cells

  • Perform real-time qPCR for cytokine genes (IL-6, TNF-α, IL-10)

  • Normalize to appropriate housekeeping genes

• Protein quantification:

  • Collect culture medium after treatment period

  • Analyze secreted cytokines via ELISA

  • Consider multiplexed assays for comprehensive cytokine profiling

Research findings have revealed that SRPK2 overexpression significantly increases both mRNA and protein levels of proinflammatory cytokines IL-6 and TNF-α while decreasing anti-inflammatory IL-10. Conversely, SRPK2 knockdown reverses this pattern, reducing proinflammatory cytokines and elevating IL-10 levels. These bidirectional changes confirm SRPK2's role in regulating the inflammatory profile of microglia .

How can researchers design experiments to elucidate the relationship between Aβ, SRPK2 expression, and microglial activation in AD models?

Designing experiments to investigate the relationship between Aβ, SRPK2 expression, and microglial activation requires a systematic approach that addresses multiple parameters:

Recommended experimental design:

• Aβ preparation and characterization:

  • Prepare Aβ42 oligomers following established protocols (dissolving in HFIP, followed by DMSO/F12 medium solution)

  • Confirm oligomer formation via immunoblotting

  • Use standardized concentrations (typically 100 μM stock solution)

• Experimental groups:

  • Control (vehicle-treated cells)

  • Aβ treatment alone

  • SRPK2 knockdown + Aβ treatment

  • SRPK2 overexpression + Aβ treatment

• Measurement parameters:

  • SRPK2 expression levels (Western blot)

  • Microglial activation markers (CD16/32, IBA-1 by immunofluorescence)

  • Cell viability (WST-1 assay)

  • Cytokine production (ELISA for IL-6, TNF-α, IL-10)

  • Cell cycle analysis (flow cytometry)

• Temporal considerations:

  • Assess immediate responses (24h)

  • Evaluate sustained effects (72h)

This design allows researchers to determine if Aβ induces SRPK2 expression, whether SRPK2 is necessary for Aβ-induced microglial activation, and if SRPK2 manipulation can alter the microglial response to Aβ. Published research has demonstrated that Aβ treatment promotes SRPK2 expression in microglial cells, and knockdown of SRPK2 can attenuate the pro-inflammatory and proliferative effects of Aβ on microglia .

What are the critical quality control parameters for validating SRPK2 Antibody, Biotin conjugated in experimental procedures?

Proper validation of SRPK2 Antibody, Biotin conjugated requires rigorous quality control steps:

Essential validation parameters:

• Specificity verification:

  • Western blot analysis showing a single band at the expected molecular weight

  • Competitive inhibition with the immunizing peptide (471-688AA of human SRPK2)

  • Comparison with alternative antibody clones

• Application-specific controls:

  • For ELISA: Standard curve generation using recombinant SRPK2

  • Positive control (human brain lysate or cells with confirmed SRPK2 expression)

  • Negative control (cells with SRPK2 knockdown)

• Technical parameters to verify:

  • Optimal working dilution determination

  • Preservation of reactivity after storage

  • Lot-to-lot consistency

• Documentation requirements:

  • Antibody specifications (host: rabbit; isotype: IgG; clonality: polyclonal)

  • Conjugation details (biotin conjugation method and ratio)

  • Purity level (should be antigen affinity purified)

The SRPK2 Antibody, Biotin conjugated from CUSABIO Technology LLC meets these critical parameters, being antigen affinity purified and specifically developed against recombinant Human SRSF protein kinase 2 protein (471-688AA). The liquid format (containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative) ensures stability for experimental applications .

How should researchers troubleshoot inconsistent results when using SRPK2 Antibody in ELISA applications?

When encountering inconsistent results with SRPK2 Antibody in ELISA applications, a systematic troubleshooting approach is recommended:

Methodological troubleshooting steps:

• Antibody-related factors:

  • Verify antibody integrity (avoid freeze-thaw cycles)

  • Confirm appropriate dilution (titrate if necessary)

  • Check biotin conjugation status (some detection systems may require enzymatic amplification)

• Sample preparation issues:

  • Ensure proper protein extraction (RIPA buffer with protease inhibitors recommended)

  • Validate protein concentration using BCA method

  • Consider sample complexity and potential interfering substances

• Protocol optimization:

  • Adjust blocking conditions (5% BSA in TBS-T is recommended)

  • Optimize incubation times and temperatures

  • Evaluate washing stringency and buffer composition

• Detection system evaluation:

  • Test alternative streptavidin-conjugated detection reagents

  • Verify signal development timing

  • Assess standard curve linearity and detection limits

• Systematic controls:

  • Include recombinant SRPK2 standards

  • Run parallel assays with known positive and negative samples

  • Consider spiking experiments to assess matrix effects

For the SRPK2 Antibody, Biotin conjugated, specific attention should be given to the buffer conditions, as the antibody is formulated with 50% Glycerol and 0.01M PBS at pH 7.4, which may affect binding kinetics in certain ELISA formats .

How can SRPK2 Antibody be utilized to investigate the Akt pathway's role in regulating SRPK2 expression in microglia?

To investigate the regulatory relationship between the Akt pathway and SRPK2 expression in microglia, researchers should implement a comprehensive experimental approach:

Recommended methodology:

• Manipulation of Akt signaling:

  • Knockdown Akt expression using targeted siRNA

  • Activate Akt using specific pathway activators

  • Combined treatments (Akt manipulation + Aβ exposure)

• Evaluation of SRPK2 expression:

  • Protein level analysis via Western blotting

  • Transcriptional analysis via qPCR

  • Subcellular localization via immunofluorescence

• Pathway verification experiments:

  • Assess phosphorylation status of Akt and downstream targets

  • Use specific pathway inhibitors to confirm mechanisms

  • Perform time-course studies to establish sequence of events

Published research has demonstrated that activation of the Akt pathway promotes SRPK2 expression in microglial cells, while Akt knockdown attenuates this effect. This relationship appears particularly relevant in the context of Aβ exposure, suggesting a potential mechanism by which Aβ induces SRPK2 expression through Akt signaling. These findings indicate that the Akt-SRPK2 axis may represent a significant therapeutic target for modulating microglial inflammatory responses in neurodegenerative conditions .

What experimental design would best evaluate the cytotoxic effects of SRPK2-modulated microglia on neuronal cells?

To evaluate how SRPK2-modulated microglia affect neuronal viability, a co-culture or conditioned media experimental design is recommended:

Detailed experimental approach:

• Microglial preparation with varied SRPK2 expression:

  • Control microglia (baseline SRPK2)

  • SRPK2-overexpressing microglia (via lentiviral vectors)

  • SRPK2-deficient microglia (via siRNA knockdown)

• Activation conditions:

  • Unstimulated

  • Aβ-stimulated (oligomeric Aβ42)

  • LPS+IFN-γ stimulated (classical inflammatory activation)

• Neuronal exposure methods:

  • Conditioned media transfer (microglial secretome exposure)

  • Trans-well co-culture (allowing for soluble factor exchange without direct contact)

  • Direct co-culture (enabling cell-cell contact interactions)

• Neuronal assessment metrics:

  • Viability assays (MTT/WST-1)

  • Apoptosis markers (TUNEL, Annexin V)

  • Morphological analysis (neurite outgrowth, branching)

  • Functional assays (calcium imaging, electrophysiology)

• Mechanistic investigations:

  • Cytokine neutralization experiments

  • Signaling pathway inhibitors

  • Transcriptome analysis of affected neurons

Research findings have demonstrated that SRPK2 deficiency significantly alleviates the cytotoxic effects of Aβ or LPS+IFN-γ exposed microglia on neuronal cells (specifically HT22 cells). This protective effect correlates with altered cytokine profiles, suggesting that SRPK2 modulation affects neuronal viability primarily through regulation of inflammatory mediator production by microglia .

How can cell cycle analysis be integrated with SRPK2 expression studies to understand microglial proliferation in neuroinflammation?

Integrating cell cycle analysis with SRPK2 expression studies provides valuable insights into the mechanisms of microglial proliferation during neuroinflammatory responses:

Integrated methodological approach:

What statistical approaches are most appropriate for analyzing SRPK2 expression data in microglial activation studies?

Recommended statistical approach:

• For comparison between two groups:

  • Student's t-test for normally distributed data

  • Mann-Whitney U test for non-normally distributed data

• For multiple group comparisons:

  • One-way ANOVA followed by post-hoc tests (Tukey's or Bonferroni) for normally distributed data

  • Kruskal-Wallis test followed by Dunn's test for non-normally distributed data

• For time-course or dose-response experiments:

  • Two-way ANOVA with time/dose and treatment as factors

  • Repeated measures ANOVA for matched samples across time points

• Data presentation guidelines:

  • Express results as mean ± SEM for three or more replicates

  • Use p-value thresholds (* p < 0.05, ** p < 0.01) for significance indication

  • Include sample size and number of independent experiments

• Correlation analysis:

  • Pearson's correlation for linear relationships between SRPK2 levels and other parameters

  • Spearman's rank correlation for non-parametric relationships

Published research on SRPK2 in microglial activation has effectively utilized Student's t-test for comparing between two experimental groups, with data presented as mean ± SEM from three independent experiments performed in triplicate. This approach provides statistical rigor while acknowledging biological variability across experimental replicates .

How should researchers interpret seemingly contradictory results between SRPK2 expression levels and different microglial activation markers?

When faced with apparently contradictory results between SRPK2 expression and microglial activation markers, researchers should employ a systematic interpretive framework:

Interpretive methodology:

• Context-dependent analysis:

  • Consider the specific activation stimulus used (Aβ vs. LPS+IFN-γ vs. IL-4)

  • Evaluate the timepoint of assessment (early vs. late responses)

  • Examine the microglial model system (primary cells vs. cell lines)

• Phenotype spectrum consideration:

  • Recognize that microglial polarization exists on a continuum rather than discrete M1/M2 states

  • Assess multiple markers across the spectrum simultaneously

  • Evaluate marker ratios rather than absolute expression levels

• Signal transduction pathway analysis:

  • Investigate potential crosstalk between SRPK2 and canonical polarization pathways

  • Examine phosphorylation states of key signaling molecules

  • Consider potential feedback mechanisms

• Resolution approach:

  • Perform time-course experiments to capture dynamic transitions

  • Utilize single-cell analysis to identify potential heterogeneous subpopulations

  • Implement genetic rescue experiments to confirm causal relationships

Research has demonstrated that while SRPK2 expression strongly correlates with M1 markers (CD16/32, IBA-1) and inversely correlates with M2 markers (CD206, Arg-1), the relationship may be complicated by the influence of Akt signaling and the specific inflammatory environment. When interpreting such data, researchers should consider that SRPK2 may differentially affect distinct aspects of microglial biology, potentially promoting certain inflammatory responses while simultaneously affecting other activation parameters through separate mechanisms .