ZFP36 Antibody, Biotin conjugated

What is ZFP36 and what is its molecular function in cellular processes?

ZFP36 (Zinc Finger Protein 36), also known as Tristetraprolin (TTP), is an AU-rich element-binding protein that functions as a post-transcriptional regulator. It promotes mRNA decay by binding to AU-rich elements in the 3′UTR of target transcripts . ZFP36 has a calculated molecular weight of 34 kDa and contains a distinguishing zinc finger structure .

Key molecular functions include:

• Suppression of inflammatory responses by targeting cytokine mRNAs

• Regulation of mRNA stability and turnover

• Interaction with transcription factors and cellular signaling pathways

ZFP36 deficiency can lead to increased inflammatory markers, particularly TNF-α, and may result in severe inflammatory syndrome .

What experimental applications are suitable for ZFP36 Antibody, Biotin conjugated?

The biotin-conjugated ZFP36 antibody can be utilized in various experimental applications with optimization for each protocol:

The biotin conjugation provides advantages for detection, purification, and complex isolation when used with streptavidin-based systems .

How should ZFP36 Antibody, Biotin conjugated be stored and handled for optimal results?

Proper storage and handling are critical for maintaining antibody functionality:

• Storage temperature: Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which can damage antibody structure and activity

• Storage buffer composition: Typically preserved in 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4

• Stability: Most antibodies remain stable for approximately 12 months from receipt date when properly stored

• Working solution preparation: Dilute only the amount needed for immediate use

• Short-term storage: 4°C for up to one week for working solutions

• Aliquoting: For antibodies requiring frequent use, prepare multiple small-volume aliquots to avoid repeated freeze-thaw cycles

What controls should be included when using ZFP36 Antibody, Biotin conjugated?

Incorporating appropriate controls is essential for experimental validity:

• Positive control: Use tissues/cells known to express ZFP36 (e.g., LPS-treated U-937 cells or RAW 264.7 cells)

• Negative control: Samples where ZFP36 expression is minimal or tissues from knockout models

• Isotype control: Rabbit IgG (for polyclonal) or Mouse IgG2a (for monoclonal) conjugated with biotin

• Blocking peptide control: Pre-incubation with immunizing peptide to demonstrate specificity

• Secondary-only control: Omit primary antibody to assess non-specific binding of detection reagents

• Knockdown validation: Compare results with ZFP36-knockdown samples to confirm specificity

How can ZFP36 Antibody, Biotin conjugated be optimized for RNA immunoprecipitation (RIP) experiments?

RNA immunoprecipitation using ZFP36 antibody enables identification of direct mRNA targets:

Methodological approach:

• Crosslinking optimization: Use 1% formaldehyde for 10 minutes at room temperature to preserve RNA-protein interactions

• Lysis buffer composition: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, and protease/phosphatase/RNase inhibitor cocktail

• Antibody amount: Typically 2-5 μg per immunoprecipitation reaction

• Biotin advantage: Use streptavidin-coated magnetic beads for efficient capture

• Washing stringency: Graduated washing steps (high salt, low salt, LiCl) to reduce background

• RNA extraction and verification: Use TRIzol reagent followed by qRT-PCR validation

Research has successfully used this approach to demonstrate direct interaction between ZFP36 and CREBBP mRNA 3′UTR, confirming ZFP36's role in regulating inflammatory responses .

What techniques can effectively validate ZFP36 binding to specific target mRNAs?

Multiple complementary approaches should be used to robustly validate ZFP36-mRNA interactions:

• Biotin-RNA pull-down assay:

  • Label RNA with biotin using Biotin-RNA Labeling Mix

  • Incubate with cell lysates containing ZFP36

  • Capture complexes with streptavidin-coated beads

  • Detect ZFP36 by Western blotting

• RIP followed by qRT-PCR:

  • Immunoprecipitate with ZFP36 antibody

  • Extract RNA from complexes

  • Perform qRT-PCR for candidate target mRNAs

• In silico analysis:

  • Identify potential AU-rich elements in 3′UTRs

  • Analyze RNA secondary structure accessibility

  • Assess binding site conservation across species

• Mutational analysis:

  • Generate constructs with mutated binding sites

  • Perform reporter assays to measure mRNA stability

  • Compare wild-type and mutant constructs

How does ZFP36 regulate inflammation in pulmonary pathologies, and what methodological approaches can investigate this function?

ZFP36 plays a critical role in regulating inflammation-associated lung damage:

Key findings:

• ZFP36 deficiency upregulates CREBBP, enhancing ischemia/reperfusion (I/R)-induced lung injury, apoptosis, and inflammation

• ZFP36 knockdown increases I/R-induced lung fibrosis through CREBBP/p53/p21/Bax pathway interactions

Methodological approaches:

• In vivo models:

  • Generate ZFP36-knockdown mice

  • Subject to intestinal I/R or hypoxia/reperfusion

  • Analyze lung tissue by Western blotting, immunohistochemistry, and real-time PCR

• In vitro cellular models:

  • Culture mouse lung epithelial (MLE-2) cells

  • Subject to hypoxia/reperfusion conditions

  • Manipulate ZFP36 expression via siRNA or overexpression

• Molecular pathway analysis:

  • Examine CREBBP expression levels

  • Assess p53/p21/Bax axis activation

  • Measure inflammatory cytokine production

  • Evaluate fibrotic markers (Twist1, Snail1)

What considerations are important when differentiating between ZFP36 and related family members (ZFP36L1, ZFP36L2) in experimental protocols?

ZFP36 belongs to a family that includes ZFP36L1 and ZFP36L2, which share structural similarities but have distinct functions:

Differentiation strategies:

• Antibody selection:

  • Verify epitope specificity to avoid cross-reactivity

  • Use antibodies raised against unique regions rather than conserved zinc finger domains

  • Validate specificity using knockout or knockdown models

• Expression pattern analysis:

  • ZFP36: Inducible by growth factors, primarily cytoplasmic

  • ZFP36L1: Contains distinctive cys-his motif, functions as nuclear transcription factor

  • ZFP36L2: Contains CSH1-type zinc fingers, binds specific 5′UUAUUUAUUU-3′ RNA sequences

• Functional validation:

  • ZFP36: Primary role in cytokine mRNA degradation

  • ZFP36L1: Regulates response to growth factors

  • ZFP36L2: Regulates growth factor-induced cellular responses

• Target specificity:

  • Perform parallel knockdown experiments

  • Use rescue experiments with specific family members

  • Identify unique mRNA targets through RIP-seq analysis

How can researchers investigate ZFP36's role in novel antiviral responses, particularly against coronaviruses?

Recent research has highlighted CCCH-type zinc finger proteins, including ZFP36, as potential antiviral factors:

Investigation methodologies:

• Viral infection models:

  • Transfect cells with ZFP36 expression vectors

  • Challenge with coronavirus (e.g., HCoV-OC43)

  • Measure viral replication by qPCR and plaque assays

• Mechanism elucidation:

  • Examine viral RNA stability in presence/absence of ZFP36

  • Investigate interaction with viral RNA using RIP or RNA pull-down

  • Assess impact on viral protein translation

• Inflammatory response assessment:

  • Measure cytokine production (TNF-α, IL-6)

  • Evaluate NF-κB pathway activation

  • Compare responses in wild-type vs. ZFP36-deficient cells

• Structure-function analysis:

  • Generate ZFP36 mutants with altered zinc finger domains

  • Assess impact on antiviral activity

  • Identify critical regions for coronavirus restriction

Research on related family member ZFP36L1 has shown significant suppression of human coronavirus OC43 replication, suggesting similar mechanisms may exist for ZFP36 .

What are common issues with ZFP36 antibody detection and their solutions?

How can researchers analyze contradictory data regarding ZFP36 function in disease models?

When faced with contradictory findings:

• Context-dependent analysis:

  • Compare tissue/cell types (different responses in epithelial vs. immune cells)

  • Evaluate disease stage (acute vs. chronic inflammation)

  • Consider microenvironment factors (cytokine milieu variations)

• Methodological reconciliation:

  • Compare antibody specificities and epitopes

  • Assess knockout/knockdown approaches (germline vs. conditional)

  • Evaluate in vitro vs. in vivo model differences

• Target gene differences:

  • ZFP36 regulates multiple mRNAs with potentially opposing effects

  • Different experimental conditions may favor regulation of different targets

  • Comprehensive transcriptome analysis may reveal dominant effects

• Pathway integration:

  • Analyze interaction with other pathways (CREBBP/p53/p21/Bax pathway)

  • Consider compensatory mechanisms by other family members

  • Evaluate feedback loops that may alter interpretation

How can ZFP36 Antibody, Biotin conjugated be used to study post-transcriptional regulation in neuroinflammatory conditions?

ZFP36 has been identified as relevant in neuroscience research areas . Methodological approach:

• Cell-type specific analysis:

  • Isolate microglia, astrocytes, and neurons

  • Compare ZFP36 expression levels across cell types

  • Evaluate response to inflammatory stimuli

• Target identification in neural cells:

  • Perform RIP using biotin-conjugated ZFP36 antibody

  • Identify neuron-specific target mRNAs

  • Validate with reporter assays

• In vivo neuroinflammation models:

  • Utilize models of stroke, traumatic brain injury, or neurodegeneration

  • Assess ZFP36 expression patterns temporally and spatially

  • Correlate with inflammatory marker expression

• Therapeutic potential assessment:

  • Modulate ZFP36 levels in neuroinflammatory models

  • Measure impact on disease progression

  • Evaluate as potential biomarker for neuroinflammatory conditions

What methodological approaches can reveal novel ZFP36 functions in autoimmune disease research?

ZFP36 has demonstrated roles in regulating autoimmune responses:

• HLA regulation analysis:

  • ZFP36 has been identified as a component of the RNP complex binding to 3′UTRs of HLA-DQA1 and HLA-DQB1 mRNAs

  • These HLA genes are associated with celiac disease susceptibility

  • ZFP36 affects mRNA stability of these autoimmune-associated genes

• Experimental approach:

  • Perform pull-down assays with biotinylated HLA riboprobes

  • Identify ZFP36 binding by Western blot

  • Analyze structural differences in CD-associated mRNAs that affect ZFP36 binding

• Clinical correlation:

  • Compare ZFP36 expression in RA patients vs. controls

  • Analyze synovial fibroblasts and macrophage subsets

  • Correlate ZFP36 levels with disease activity markers

• Integration with other immune regulators:

  • Assess interaction with NF-κB pathway

  • Evaluate impact on T cell activation

  • Analyze cytokine network regulation