ZHX1-C8orf76 Antibody

What is ZHX1-C8orf76 and why is it significant in research?

ZHX1-C8orf76 represents a naturally occurring readthrough transcript between the neighboring zinc fingers and homeoboxes 1 (ZHX1) and chromosome 8 open reading frame 76 (C8orf76) genes. The readthrough transcript encodes a protein that shares sequence identity with the downstream gene but possesses a distinct N-terminus encoded by the upstream gene's exon structure. ZHX1 functions as a transcriptional repressor involved in crucial biological processes including cell proliferation and differentiation, while C8orf76 remains relatively poorly characterized with potential roles in cell signaling and metabolism .

This readthrough phenomenon represents an important area of research in gene regulation and protein diversity. The protein has a molecular weight of approximately 98,098 Da and plays potential roles in transcriptional regulation networks that remain to be fully elucidated . Understanding ZHX1-C8orf76 function and expression has implications for both basic cellular biology and potential disease mechanisms.

What types of ZHX1-C8orf76 antibodies are currently available for research applications?

Several configurations of ZHX1-C8orf76 antibodies have been developed for research applications, with varying conjugations and host species:

The most extensively validated antibody appears to be the rabbit polyclonal (PACO40158), which has demonstrated efficacy in Western blotting, immunohistochemistry, and ELISA applications. This antibody has been tested against both human and mouse samples, making it versatile for comparative studies . For specialized applications requiring biotin conjugation, options such as abx310481 are available for techniques requiring signal amplification or avidin-biotin detection systems .

What are the recommended protocols for Western blotting with ZHX1-C8orf76 antibody?

For Western blotting applications using ZHX1-C8orf76 antibody, the following methodological approach is recommended:

Sample Preparation:

• Prepare tissue lysates in standard RIPA buffer supplemented with protease inhibitors

• Load 20-40 μg of total protein per lane for cell lysates or 40-60 μg for tissue extracts

• Include positive controls such as mouse heart or kidney tissue, which have demonstrated detectable expression of the target protein

Primary Antibody Protocol:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Dilute ZHX1-C8orf76 antibody at 1:500-1:2000 in blocking buffer (optimal concentration determined through validation)

• Incubate membrane with primary antibody solution overnight at 4°C with gentle agitation

• Wash membrane 3-5 times with TBST, 5 minutes per wash

Detection:

• Use secondary antibody (goat anti-rabbit IgG-HRP) at 1:10000 dilution

• Expected band size: 33 kDa (observed in validated tissues)

• For enhanced specificity, pre-absorption with immunizing peptide can verify antibody specificity

Researchers should note that a distinct 33 kDa band has been observed in mouse heart and kidney tissues, suggesting specific detection of the target protein .

What are the optimal conditions for immunohistochemistry using ZHX1-C8orf76 antibody?

For immunohistochemistry applications, ZHX1-C8orf76 antibody has been validated on human tissues including tonsil and cervical cancer samples . The following protocol is recommended:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin

• Process and embed in paraffin following standard histological procedures

• Section tissues at 4-6 μm thickness

• Mount sections on positively charged slides

Staining Protocol:

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) for 15-20 minutes

• Block endogenous peroxidase with 3% H₂O₂ in methanol for 10 minutes

• Apply protein block (5% normal goat serum) for 30 minutes

• Dilute ZHX1-C8orf76 antibody at 1:20-1:200 (with 1:100 being optimal for most applications)

• Incubate sections with primary antibody overnight at 4°C

• Apply appropriate detection system (HRP-polymer or biotin-streptavidin)

• Develop with DAB and counterstain with hematoxylin

Validated Tissues:
The antibody has demonstrated specific staining in human tonsil tissue and cervical cancer samples, making these appropriate positive controls for validating the protocol in your laboratory .

How can ZHX1-C8orf76 antibody be integrated with CRISPR-Cas9 gene editing studies?

Integrating ZHX1-C8orf76 antibody detection with CRISPR-Cas9 gene editing provides powerful approaches for functional studies:

CRISPR Knockout Validation:

• Design guide RNAs targeting the ZHX1-C8orf76 gene using validated sequences (such as those designed by the Zhang laboratory)

• Transfect cells with Cas9 and gRNA expression constructs

• Validate knockout efficiency using ZHX1-C8orf76 antibody by Western blotting at 1:500-1:2000 dilution

• Compare protein levels between wild-type and edited cells

• Verify specificity using at least two different guide RNAs to control for off-target effects

Methodological Considerations:

• When selecting guide RNAs, consider targeting conserved exons present in both the individual gene and the readthrough transcript

• The Zhang laboratory has designed specific guide RNA sequences that uniquely target ZHX1-C8orf76 with minimal off-target risk

• Use at least two guide RNA constructs per gene to increase success likelihood

• Verify guide RNA sequences against your specific target sequence before proceeding

This integrated approach allows researchers to correlate phenotypic changes with protein expression levels and localization patterns in knockout or knock-in models.

What methods can be used to investigate protein-protein interactions involving ZHX1-C8orf76?

To characterize the interactome of ZHX1-C8orf76, several methodological approaches can be employed:

Co-Immunoprecipitation (Co-IP):

• Prepare cell or tissue lysates under non-denaturing conditions

• Pre-clear lysate with protein G beads

• Incubate pre-cleared lysate with ZHX1-C8orf76 antibody (2-5 μg per mg of total protein)

• Capture antibody-protein complexes with protein G beads

• Wash extensively to remove non-specific interactions

• Elute bound proteins and analyze by Western blotting for suspected interaction partners

Proximity Ligation Assay (PLA):

• Prepare fixed cells or tissue sections

• Incubate with ZHX1-C8orf76 antibody (1:100) and antibody against suspected interaction partner

• Apply secondary antibodies conjugated to oligonucleotides

• Perform ligation and amplification steps

• Visualize interaction signals by fluorescence microscopy

Bimolecular Fluorescence Complementation (BiFC):

• Create fusion constructs of ZHX1-C8orf76 and potential partners with split fluorescent protein fragments

• Co-transfect constructs into appropriate cell lines

• Analyze fluorescence reconstitution indicative of protein proximity

• Validate interactions using co-immunoprecipitation with ZHX1-C8orf76 antibody

These approaches should be combined for comprehensive characterization of protein interactions, with antibody-based methods providing verification of results from fluorescence-based techniques.

What are common technical challenges when working with ZHX1-C8orf76 antibody and how can they be resolved?

Researchers may encounter several challenges when working with ZHX1-C8orf76 antibody:

For challenging applications, consider:

• Using freshly prepared samples and buffers

• Including appropriate positive controls (mouse heart/kidney for Western blot, human tonsil for IHC)

• Performing antibody validation with immunizing peptide competition

• Testing multiple fixation and antigen retrieval protocols for IHC applications

Researchers should note that the ZHX1-C8orf76 antibody shows optimal results when used at a dilution of 1:500-1:2000 for Western blotting and 1:20-1:200 for IHC applications .

How can specificity be ensured when detecting ZHX1-C8orf76 in complex samples?

Ensuring specificity when working with ZHX1-C8orf76 antibody requires rigorous methodological approaches:

Antibody Validation Strategies:

• Genetic Controls: Compare antibody signal between wild-type samples and those with CRISPR-mediated knockout of ZHX1-C8orf76

• Peptide Competition: Pre-incubate antibody with immunizing peptide to block specific binding sites

• Multiple Antibodies: Validate results using antibodies targeting different epitopes of the same protein

• Expression System Controls: Compare endogenous protein detection with samples overexpressing recombinant ZHX1-C8orf76 protein

Technical Approaches for Enhanced Specificity:

• Optimized Blocking: Use 5% BSA or commercial blocking reagents specifically designed to reduce non-specific binding

• Titration Series: Perform antibody dilution series to identify optimal concentration (1:500-1:2000 for WB, 1:20-1:200 for IHC)

• Extended Washing: Increase number and duration of washes to reduce background

• Sample Preparation: Ensure complete denaturation for Western blotting applications

• Dual Detection: Confirm results using orthogonal methods (e.g., mass spectrometry validation of immunoprecipitated proteins)

When working with human samples, researchers should be aware that the antibody has been validated for reactivity with human and mouse tissues, with specific detection demonstrated in both species .

What are the emerging research areas utilizing ZHX1-C8orf76 antibodies?

ZHX1-C8orf76 antibodies are being utilized in several emerging research areas:

Transcriptional Regulation Studies:
The ZHX1 component functions as a transcriptional repressor involved in cell proliferation and differentiation pathways . Researchers are using these antibodies to elucidate how the readthrough transcript might modify or extend this regulatory function.

Cancer Biology Applications:
ZHX1-C8orf76 antibodies have been validated in cervical cancer tissues , suggesting potential roles in oncology research. The antibody can be used to:

• Compare expression levels between normal and malignant tissues

• Correlate expression with clinical parameters and outcomes

• Investigate potential roles in cancer cell signaling networks

Readthrough Transcript Biology:
As a naturally occurring readthrough transcript, ZHX1-C8orf76 provides a model system for studying this important mechanism of gene expression regulation and protein diversity. Researchers are using the antibody to:

• Map tissue-specific expression patterns of the readthrough protein

• Compare expression of individual genes versus the readthrough product

• Investigate regulatory mechanisms controlling readthrough frequency

Future Research Directions:

• Integration with multi-omics approaches for comprehensive functional characterization

• Development of conditional knockout models to study tissue-specific functions

• Investigation of potential biomarker applications in disease states

• Comparative studies across species to understand evolutionary conservation

How can ZHX1-C8orf76 antibodies be integrated into multi-omics research approaches?

Integration of ZHX1-C8orf76 antibodies into multi-omics research frameworks represents an emerging frontier:

Proteogenomic Integration:

• Combine CRISPR-Cas9 genomic editing of ZHX1-C8orf76 with antibody-based protein detection to correlate genotype with protein expression

• Validate RNA-seq findings of alternate splicing or readthrough events at the protein level using the antibody

• Correlate protein expression with genomic alterations in disease states

Protein-Protein Interaction Networks:

• Use immunoprecipitation with ZHX1-C8orf76 antibody followed by mass spectrometry to map the interactome

• Validate high-confidence interactions with co-immunoprecipitation and proximity ligation assays

• Integrate interaction data with transcriptomic profiles to identify coordinated regulatory networks

Spatial Proteomics Applications:

• Employ immunohistochemistry with ZHX1-C8orf76 antibody (1:20-1:200 dilution) to map tissue and subcellular localization

• Combine with multiplexed immunofluorescence to analyze co-localization with interaction partners

• Correlate spatial distribution with functional data from genetic perturbation experiments

Methodological Integration Framework:
Graph convolutional networks and other computational approaches can integrate multi-omics data including antibody-based protein detection to identify potential driver genes in cancer and other diseases . This approach enables researchers to:

• Correlate protein expression detected by ZHX1-C8orf76 antibody with transcriptomic and genomic data

• Identify regulatory relationships and network connections

• Develop predictive models of protein function in normal and disease states