CSRNP3 Antibody, HRP conjugated

What is CSRNP3 and why is it important in research?

CSRNP3 (cysteine-serine-rich nuclear protein 3) is a protein-coding gene also known as MBU1, TAIP2, TAIP-2, PPP1R73, and FAM130A2 . It is predicted to enable DNA-binding transcription factor activity specific to RNA polymerase II and sequence-specific DNA binding activity. CSRNP3 is thought to be involved in positive regulation of apoptotic processes and transcription by RNA polymerase II . The protein is primarily localized to chromatin within the nucleus . Research into CSRNP3 is particularly relevant to studies involving transcriptional regulation and has been linked to longevity studies, as indicated by genome-wide association meta-analyses of human longevity .

What are the common applications for CSRNP3 antibodies with HRP conjugation?

CSRNP3 antibodies with HRP conjugation are primarily utilized in the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Typically used at dilutions of 1:1000

• Western Blot: Typically used at dilutions of 1:100-500

• Immunohistochemistry: For detecting CSRNP3 in fixed tissue samples

• Immunocytochemistry: For subcellular localization studies

The HRP conjugation enables direct detection without the need for secondary antibodies, streamlining experimental workflows and potentially reducing background signal.

What are the optimal conditions for using HRP-conjugated CSRNP3 antibodies in Western blot applications?

For optimal Western blot results with HRP-conjugated CSRNP3 antibodies:

• Sample preparation: Use RIPA or NP-40 based lysis buffers supplemented with protease inhibitors

• Loading amount: 15-20 μg of total protein lysate per lane is typically sufficient

• Dilution ratio: Use at 1:100-500 dilution in 5% non-fat dry milk (NFDM) in TBST

• Incubation time and temperature: Incubate for 1 hour at room temperature or overnight at 4°C

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Washing: Three 5-minute washes with TBST after antibody incubation

• Detection method: Enhanced chemiluminescence (ECL) with exposure times ranging from 30 seconds to 5 minutes

For nuclear proteins like CSRNP3, ensure proper nuclear extraction protocols are followed to maximize yield and maintain protein integrity.

How should CSRNP3 antibody, HRP conjugated be stored to maintain optimal activity?

To maintain the activity of HRP-conjugated CSRNP3 antibodies:

• Storage temperature: Store at -20°C for long-term storage

• Working aliquots: Keep at 4°C for up to three months

• Buffer conditions: Store in PBS with stabilizers (often containing 50% glycerol, 0.05% preservative, and 0.5% BSA at pH 7.3)

• Light sensitivity: Protect from prolonged light exposure, as HRP conjugates can be light-sensitive

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles; make small working aliquots from stock

• Stabilization: Some antibody preparations include stabilizers like proclin300 to maintain activity

Improper storage can lead to decreased signal intensity, increased background, and potentially false-negative results.

How can specificity of CSRNP3 antibody be validated in experimental systems?

Validating CSRNP3 antibody specificity is critical for reliable experimental outcomes. A comprehensive validation approach includes:

• Western blot analysis: Look for a band of expected molecular weight (~26 kDa theoretical, may run higher due to post-translational modifications)

• Knockout/knockdown controls:

  • Use CSRNP3 knockout tissues/cells as negative controls

  • siRNA/shRNA knockdown samples should show reduced signal intensity

• Immunoprecipitation followed by mass spectrometry:

  • Confirm pulled-down protein identity through peptide sequencing

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide (N-terminal epitope for the antibody in search result )

  • Signal should be decreased or abolished

• Cross-reactivity testing:

  • Test against related family members (CSRNP1, CSRNP2) to ensure specificity

  • Particularly important as CSRNP proteins share structural similarities

• Immunofluorescence co-localization:

  • Co-stain with known nuclear markers to confirm expected nuclear localization

Thorough validation prevents misleading interpretations of experimental data and ensures reproducibility across research groups.

What potential cross-reactivity issues might arise with CSRNP3 antibodies and how can they be addressed?

Potential cross-reactivity issues with CSRNP3 antibodies include:

• Family member cross-reactivity: CSRNP3 belongs to a family that includes CSRNP1 and CSRNP2, which share conserved domains

• Isoform specificity: Multiple isoforms of CSRNP3 may exist; the antibody in search result was raised against the N-terminal region, which may not detect all isoforms

• Non-specific binding to other cysteine-serine-rich proteins: Structural similarities may cause cross-reactivity

To address these issues:

• Pre-adsorption: Pre-incubate antibody with recombinant CSRNP1/CSRNP2 to remove cross-reactive antibodies

• Multiple antibody approach: Use antibodies targeting different epitopes of CSRNP3

• Western blot analysis in different cell types: Compare band patterns across cell lines with known CSRNP3 expression levels

• Careful optimization of antibody dilution: Higher dilutions often reduce non-specific binding while maintaining specific signal

• Extended blocking: Increase blocking time with 5% BSA or non-fat dry milk to reduce non-specific interactions

What are common causes of high background when using HRP-conjugated CSRNP3 antibodies and how can they be resolved?

High background when using HRP-conjugated CSRNP3 antibodies can be caused by several factors:

• Insufficient blocking:

  • Solution: Extend blocking time to 2 hours or overnight and increase blocker concentration to 5-10%

• Antibody concentration too high:

  • Solution: Titrate antibody from 1:1000 to 1:10000 to determine optimal concentration

• Insufficient washing:

  • Solution: Increase number of washes (5-6 times) and duration (10 minutes each)

• HRP conjugate degradation:

  • Solution: Use fresh antibody aliquots and verify proper storage conditions

• Non-specific binding to membrane:

  • Solution: Add 0.1-0.5% Tween-20 to blocking and washing buffers

• Sample overloading:

  • Solution: Reduce protein amount to 10-15 μg per lane

• Detector sensitivity too high:

  • Solution: Reduce exposure time when using ECL detection

A systematic approach to troubleshooting, changing one parameter at a time, will help identify the specific cause of high background.

How can signal strength be optimized when CSRNP3 expression is low in experimental samples?

When CSRNP3 expression is low, several strategies can enhance signal strength:

• Sample enrichment:

  • Perform nuclear extraction to concentrate CSRNP3

  • Use immunoprecipitation to enrich the target protein before Western blot

• Signal amplification:

  • Utilize tyramide signal amplification (TSA) which can enhance HRP signal by up to 100-fold

  • Use enhanced chemiluminescent substrates with higher sensitivity

• Blocking optimization:

  • Test different blocking agents (BSA, casein, commercial blockers) to find optimal signal-to-noise ratio

• Extended primary antibody incubation:

  • Incubate at 4°C for 24-48 hours to maximize binding

• Antibody concentration adjustment:

  • Use higher concentration of primary antibody within recommended range

  • For Western blot, try 1:100 dilution instead of 1:500

• Membrane optimization:

  • Use PVDF membranes with higher protein binding capacity

  • Reduce pore size to 0.22 μm to prevent protein loss

• Detection system enhancement:

  • Use digital imaging systems with integration capability to collect signal over time

A combination of these approaches can significantly improve detection of low-abundance CSRNP3.

How can CSRNP3 antibodies be used to investigate its role in transcriptional regulation?

CSRNP3 is predicted to function as a transcription factor . To investigate its role in transcriptional regulation:

• Chromatin Immunoprecipitation (ChIP):

  • Use CSRNP3 antibodies to immunoprecipitate chromatin

  • Sequence bound DNA (ChIP-seq) to identify genomic binding sites

  • Combine with RNA-seq to correlate binding with gene expression changes

• Transcriptional reporter assays:

  • Similar to studies with other CSRNP family members, design GAL4 fusion proteins to assess transcriptional activity

  • CSRNP-1 showed 70-fold higher reporter activity than control constructs

• Co-immunoprecipitation:

  • Identify protein interaction partners involved in transcriptional complexes

  • Western blot with HRP-conjugated CSRNP3 antibody can confirm CSRNP3 presence in complexes

• Nuclear localization studies:

  • Use immunofluorescence to track CSRNP3 localization in response to stimuli

  • Confirm nuclear localization with nuclear markers

• DNA-binding assays:

  • Electrophoretic mobility shift assay (EMSA) with purified CSRNP3 and candidate DNA sequences

  • Use antibody for supershift assays to confirm specificity

Note that while CSRNP-1 showed strong transactivation activity in 293T cells, CSRNP-2 and CSRNP-3 did not show activity in these cells but did show transactivation in yeast reporter strains .

What controls should be included when using CSRNP3 antibodies in experiments related to longevity studies?

When investigating CSRNP3 in longevity studies, as suggested by its association with human longevity in genome-wide studies , include these controls:

• Age-matched tissue/cell samples:

  • Compare CSRNP3 expression across different age groups

  • Include samples from exceptionally long-lived individuals and average lifespan individuals

• Genetic controls:

  • Include samples with known longevity-associated variants in CSRNP3

  • Compare individuals with and without the specific variants identified in genome-wide association studies

• Tissue-specific expression controls:

  • Analyze CSRNP3 expression in multiple tissues relevant to aging

  • Include both high-expressing and low-expressing tissues as controls

• Pathway controls:

  • Measure expression of known longevity-associated genes (e.g., FOXO family)

  • Assess correlation between CSRNP3 and established longevity markers

• Intervention controls:

  • Compare CSRNP3 expression before and after longevity-promoting interventions

  • Include samples from caloric restriction or other lifespan-extending protocols

• Species comparisons:

  • Compare CSRNP3 expression and function across species with different lifespans

  • Use cross-reactive antibodies or species-specific antibodies for comparative studies

Proper controls are essential for establishing causative relationships between CSRNP3 and longevity phenotypes rather than merely correlative associations.

How can HRP-conjugated CSRNP3 antibodies be integrated into multiplexed detection systems?

Integrating HRP-conjugated CSRNP3 antibodies into multiplexed detection systems requires careful planning:

• Sequential detection strategies:

  • Perform HRP detection first

  • Inactivate HRP using sodium azide or hydrogen peroxide before subsequent staining

  • Re-block the membrane/slide before adding the next antibody

• Spectral separation approaches:

  • Use HRP-conjugated CSRNP3 antibody with one chromogenic substrate (e.g., DAB)

  • Use alkaline phosphatase-conjugated antibodies for other targets with different colorimetric substrates (e.g., BCIP/NBT)

• Tyramide signal amplification with different fluorophores:

  • Use HRP to deposit specific tyramide-conjugated fluorophores

  • Inactivate HRP before repeating with different antibody and fluorophore combinations

• Different antibody host species:

  • Use rabbit-derived CSRNP3 antibody alongside mouse or goat antibodies for other targets

  • Detect with species-specific secondary antibodies

• Microfluidic approaches:

  • Sequential staining in microfluidic devices with washing/stripping steps between detections

A systematic optimization approach is necessary to ensure each antibody functions properly within the multiplexed system without cross-interference.

Can HRP-conjugated CSRNP3 antibodies be used effectively in flow cytometry applications?

While HRP-conjugated antibodies are not conventional choices for flow cytometry, they can be adapted with specific considerations:

• Signal conversion requirements:

  • HRP requires a chromogenic or fluorogenic substrate to generate detectable signal

  • Use substrates like dihydrorhodamine 123 that become fluorescent when oxidized by HRP

• Protocol adaptation:

  • Fix and permeabilize cells thoroughly for nuclear antigen access

  • Include membrane permeabilization steps to allow enzyme substrates to enter cells

  • Use lower antibody concentrations (1:500-1:1000) to minimize background

• Signal amplification considerations:

  • Tyramide signal amplification can be used to deposit fluorophores in cells

  • This creates a permanent fluorescent signal detectable by flow cytometry

• Technical limitations:

  • Lower sensitivity compared to direct fluorophore conjugates

  • Additional washing steps required compared to traditional protocols

  • Potential for cell aggregation during enzymatic reaction

• Controls and alternatives:

  • Include unstained, isotype-HRP, and known positive controls

  • Consider using unconjugated primary with fluorophore-conjugated secondary if sensitivity issues arise

For most flow cytometry applications, it is generally preferable to use directly fluorophore-conjugated antibodies rather than HRP conjugates, but the above adaptations can make HRP-conjugated antibodies functional if necessary.

How does CSRNP3 function compare to other family members (CSRNP1, CSRNP2) in research applications?

The CSRNP family members have distinct functional characteristics that influence experimental approaches:

Research applications should consider these differences:

• Transcriptional studies:

  • CSRNP1 is preferable for mammalian cell-based reporter assays

  • CSRNP3 may require yeast systems or specialized mammalian contexts to observe activity

• Domain analysis:

  • Functional domains are better characterized in CSRNP1

  • CSRNP3 studies should focus on determining if functional domains are conserved

• Cellular context:

  • Cell type selection is critical as CSRNP3 activity appears highly context-dependent

  • Consider testing multiple cell lines to find appropriate experimental systems

• Protein interaction networks:

  • CSRNP family members likely have distinct interaction partners

  • Co-immunoprecipitation studies should be designed to identify unique CSRNP3 interactors

Understanding these differences is essential for designing appropriate experiments and interpreting results correctly.

What are the emerging research applications for studying CSRNP3 in relation to human disease?

Emerging research applications for CSRNP3 include:

• Longevity and aging research:

  • CSRNP3 has been identified in genome-wide association meta-analyses of human longevity

  • Applications include:

    • Genetic screening for CSRNP3 variants in longevity cohorts

    • Assessment of CSRNP3 expression changes during aging

    • Investigation of CSRNP3-regulated genes in age-related pathways

• Apoptosis regulation:

  • CSRNP3 is predicted to be involved in positive regulation of apoptotic processes

  • Applications include:

    • Measuring CSRNP3 expression in response to apoptotic stimuli

    • Assessing impact of CSRNP3 knockdown/overexpression on cell survival

    • Identifying apoptotic genes regulated by CSRNP3

• Transcriptional regulation in disease contexts:

  • As a predicted DNA-binding transcription factor , CSRNP3 may regulate disease-relevant genes

  • Applications include:

    • ChIP-seq to identify disease-relevant transcriptional targets

    • Expression analysis in disease tissues

    • Correlation of CSRNP3 activity with disease progression markers

• Potential therapeutic targeting:

  • If validated as a disease modifier, CSRNP3 could be targeted therapeutically

  • Applications include:

    • High-throughput screening for compounds modulating CSRNP3 expression/activity

    • Development of peptide inhibitors targeting CSRNP3 protein interactions

    • CRISPR-based approaches to modulate CSRNP3 in disease models

Each application requires careful experimental design and appropriate controls to establish the biological significance of CSRNP3 in disease contexts.