purb Antibody

What is PURB and what cellular functions does it participate in?

PURB (purine-rich element binding protein B) is a 33-40 kDa protein involved in transcriptional regulation. It belongs to the family of single-stranded DNA-binding proteins that interact with purine-rich sequences. PURB functions primarily as a transcriptional regulator that can form complexes with various nucleic acids and proteins to control gene expression . Recent research has demonstrated PURB's involvement in myogenesis (muscle development) and hepatic glucose production. Additionally, PURB has been found to participate in the regulation of circular RNAs, forming heterotypic complexes that can inhibit host gene transcription .

What experimental applications are PURB antibodies validated for?

PURB antibodies have been validated for multiple research applications with varying dilution requirements for optimal results:

Researchers should note that optimal dilutions may be sample-dependent, and titration is recommended for each experimental system to obtain optimal results .

What is the typical molecular weight observed for PURB in Western blot analysis?

When using PURB antibodies in Western blot applications, the protein typically appears at 33-40 kDa, which aligns with its calculated molecular weight of 33 kDa (312 amino acids) . Variations in apparent molecular weight may occur due to post-translational modifications, splice variants, or sample preparation methods. When performing Western blot analysis with PURB antibodies, it is advisable to include positive controls such as C2C12 cells, HeLa cells, or mouse liver tissue, as these have been validated to express detectable levels of PURB protein .

What species reactivity is available for commercial PURB antibodies?

PURB antibodies have been tested and validated for reactivity with multiple species:

When selecting a PURB antibody for your research, ensure the antibody has been validated in your species of interest. Cross-reactivity information is typically provided in product documentation, and sequence homology analyses can help predict potential reactivity in species not explicitly tested .

How can PURB antibodies be effectively used in ChIP experiments to study transcriptional regulation?

For chromatin immunoprecipitation (ChIP) experiments with PURB antibodies, researchers should follow this optimized protocol:

• Crosslink cells with 1% formaldehyde for 10 minutes at room temperature.

• Quench the crosslinking reaction with glycine (final concentration 0.125M) for 5 minutes.

• Lyse cells and fragment chromatin using enzymatic digestion or sonication to obtain fragments of 200-500 bp.

• Dilute chromatin and immunoprecipitate with PURB antibody (typically 2-5 μg) overnight at 4°C.

• Add protein G magnetic beads and incubate for 2 hours at 4°C.

• Perform sequential washes with low-salt and high-salt buffers to reduce non-specific binding.

• Elute DNA-protein complexes and reverse crosslinks.

• Purify DNA for subsequent qPCR analysis.

The percentage input method is recommended for data analysis, where the signal from each immunoprecipitated sample is normalized to the input chromatin . This approach has successfully identified PURB binding to promoter regions, such as the TTN gene promoter in myogenic cells .

What protocols are recommended for RNA immunoprecipitation (RIP) with PURB antibodies?

For RNA immunoprecipitation experiments investigating PURB-RNA interactions:

• Lyse cells in an appropriate buffer containing RNase inhibitors and remove DNA.

• Set aside 10% of the lysate as input control.

• Divide the remaining lysate for immunoprecipitation with PURB antibody (Proteintech) and control IgG antibody.

• Incubate samples with the respective antibodies at 4°C for 16 hours with gentle rotation.

• Add pre-balanced protein A/G beads and incubate for an additional hour at 4°C.

• Wash the beads thoroughly to remove non-specific binding.

• Elute RNA using an elution buffer containing proteinase K.

• Extract RNA using phenol-chloroform-isoamyl alcohol mixture.

• Analyze the precipitated RNA by qRT-PCR, calculating enrichment using the percentage input method.

This approach has successfully demonstrated interactions between PURB protein and circular RNAs such as circTTN , providing insights into novel regulatory mechanisms involving PURB.

How do researchers validate the specificity of PURB antibodies in experimental systems?

Validating antibody specificity is critical for reliable research results. For PURB antibodies, multiple validation strategies should be employed:

• CRISPR/Cas9 Knockout Validation: Use CRISPR guide RNAs targeting the PURB gene to generate knockout cell lines. The absence of signal in knockout samples confirms antibody specificity. Multiple gRNA constructs are recommended to increase success probability .

• siRNA Knockdown: Transfect cells with PURB-specific siRNAs and confirm reduced signal in Western blot or immunofluorescence applications. The search results indicate this approach has been successfully used to validate PURB antibody specificity .

• Recombinant Protein Controls: Use purified recombinant PURB protein as a positive control in Western blot applications.

• Cross-validation with Multiple Antibodies: Compare results using antibodies targeting different epitopes of PURB.

• Mass Spectrometry Confirmation: For immunoprecipitation experiments, verify pulled-down proteins by mass spectrometry to confirm PURB presence.

The literature shows at least 2 publications have utilized knockdown/knockout approaches to validate PURB antibodies, and 9 publications have successfully used these antibodies in Western blot applications .

What is the relationship between PURB and circular RNAs, and how can this be studied?

Recent research has revealed an important regulatory relationship between PURB and circular RNAs, particularly circTTN. This relationship can be investigated using several techniques:

• RNA Pull-down Assays: Biotin-labeled circRNA probes can be used to pull down associated proteins, followed by Western blot detection of PURB. Research has shown that circTTN can physically interact with PURB protein .

• RIP Analysis: Using PURB antibodies for RNA immunoprecipitation can identify associated circular RNAs. Studies have demonstrated that PURB protein interacts with circTTN .

• Functional Studies: Knockdown of PURB combined with circRNA overexpression or knockdown experiments can reveal their functional relationships. Research indicates that circTTN inhibits the transcription and myogenesis of the host gene TTN by recruiting PURB proteins to form heterotypic complexes .

• ChIP-qPCR: This technique can determine whether PURB binds to the promoter regions of genes encoding circular RNAs or their host genes, providing insights into regulatory mechanisms.

These methodologies can help researchers understand the complex regulatory networks involving PURB and circular RNAs in processes like myogenesis and transcriptional regulation.

What are common issues researchers encounter with PURB antibodies and how can they be resolved?

When working with PURB antibodies, researchers may encounter several challenges:

For optimal Western blot results, follow the specific protocol provided for PURB antibody (e.g., 18128-1-AP) which includes detailed instructions for sample preparation, blotting conditions, and detection methods .

How do experimental conditions affect PURB antibody performance in different applications?

The performance of PURB antibodies can be significantly influenced by experimental conditions:

• Fixation Methods for Immunofluorescence: For PURB detection in cell samples, 4% paraformaldehyde fixation for 15 minutes followed by 0.1% Triton X-100 permeabilization typically yields optimal results. Methanol fixation may alter epitope accessibility.

• Buffer Systems for Western Blot: RIPA buffer supplemented with protease inhibitors is generally effective for PURB extraction. For nuclear proteins like PURB, additional nuclear extraction steps may improve yield.

• Blocking Agents: 5% non-fat milk in TBST is typically effective for blocking in Western blot applications, while 5% BSA may be preferable for phospho-specific applications or when using biotin-streptavidin systems.

• Incubation Temperature and Time: Primary antibody incubation at 4°C overnight generally provides superior results compared to shorter incubations at room temperature.

• Sample Types: PURB antibodies have been validated in various sample types including C2C12 cells, mouse liver tissue, and HeLa cells . Cell-specific expression levels may necessitate adjustment of experimental parameters.

How can researchers optimize PURB antibody use in co-immunoprecipitation experiments?

For successful co-immunoprecipitation (co-IP) of PURB and its interacting partners:

• Lysis Conditions: Use gentle lysis buffers (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA with protease inhibitors) to preserve protein-protein interactions.

• Antibody Amount: For PURB immunoprecipitation, use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate .

• Pre-clearing Step: Pre-clear lysates with protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Controls: Always include an isotype-matched IgG control to identify non-specific interactions.

• Crosslinking Consideration: For weak or transient interactions, consider using chemical crosslinkers like DSP (dithiobis(succinimidyl propionate)) before lysis.

• Elution Conditions: For mass spectrometry analysis of PURB complexes, elute under conditions that minimize antibody contamination, such as using competitive peptide elution.

• Verification: Confirm successful immunoprecipitation by probing a small portion of the IP samples for PURB before proceeding to identification of interacting partners.

These optimized methods have successfully identified PURB interactions with various proteins and nucleic acids, including circular RNAs like circTTN .

How is PURB involved in muscle development and what research techniques best elucidate this function?

PURB plays a significant role in muscle development (myogenesis), with research showing its involvement in regulatory pathways affecting muscle cell proliferation and differentiation:

• Expression Analysis: Western blot analysis shows PURB protein expression changes during natural differentiation of C2C12 myoblasts, suggesting a regulatory role in myogenesis .

• Loss-of-Function Studies: siRNA-mediated knockdown of PURB affects proliferation and differentiation of C2C12 cells, providing direct evidence of its functional importance in muscle development .

• Mechanistic Studies: PURB forms protein-RNA complexes with circular RNAs like circTTN to regulate transcription of the host gene TTN, which is essential for muscle function .

• ChIP-qPCR Analysis: This technique has revealed PURB binding to promoter regions of muscle-related genes, providing insights into its transcriptional regulatory functions .

• Histological Assessment: Immunofluorescence staining of muscle tissue sections using PURB antibodies can reveal spatial distribution and expression patterns during different developmental stages.

These approaches collectively provide comprehensive insights into PURB's role in muscle biology and development, with particular relevance to understanding myogenic disorders and potential therapeutic interventions.

What emerging applications are being developed for PURB antibodies in research?

Recent scientific advances have expanded the applications of PURB antibodies beyond traditional uses:

• Single-Cell Techniques: Integration of PURB antibodies in single-cell Western blot or CyTOF applications to understand cell-to-cell variability in PURB expression and function.

• Proximity Ligation Assays (PLA): Combining PURB antibodies with antibodies against predicted interacting partners to visualize and quantify protein-protein interactions in situ.

• CRISPR Screens: Using PURB antibodies to validate phenotypes in CRISPR-based functional genomics screens targeting PURB-related pathways .

• Circular RNA Research: PURB antibodies are increasingly being used to understand the emerging role of PURB in circular RNA regulation, an expanding field in RNA biology .

• Neurodegenerative Disease Research: Investigating PURB's potential roles in neurodegenerative conditions through immunohistochemical analysis of patient samples.

• Metabolic Research: Recent findings suggest PURB's involvement in hepatic glucose production, opening new research directions in metabolic disorders .

These emerging applications demonstrate the versatility of PURB antibodies in addressing complex biological questions across multiple research domains.

How do post-translational modifications affect PURB function and antibody detection?

Post-translational modifications (PTMs) can significantly impact both PURB protein function and its detection by antibodies:

• Common PTMs: PURB undergoes several post-translational modifications including phosphorylation, which can regulate its DNA/RNA binding capacity and protein-protein interactions.

• Detection Challenges: PTMs may alter epitope accessibility or recognition, potentially affecting antibody binding. This explains why PURB is sometimes detected as multiple bands or at slightly different molecular weights (observed range of 33-40 kDa despite a calculated mass of 33 kDa) .

• Modification-Specific Antibodies: Currently, most available PURB antibodies target total protein rather than specific modified forms. Researchers investigating particular PTMs may need to use general PURB immunoprecipitation followed by PTM-specific detection methods.

• Sample Preparation Considerations: Phosphatase inhibitors should be included in lysis buffers when studying phosphorylated forms of PURB. Similarly, deubiquitinase inhibitors may be necessary when investigating ubiquitination.

• Functional Implications: Different PTMs may direct PURB to distinct subcellular compartments or protein complexes. Researchers should consider subcellular fractionation followed by Western blot analysis to determine compartment-specific distributions.

Understanding these modifications is essential for comprehensive characterization of PURB function in various cellular contexts and may explain seemingly contradictory results across different experimental systems.