zgc:56556 Antibody

What is zgc:56556 protein and what is its significance in zebrafish models?

zgc:56556 (Entrez Gene ID: 393302, UniProt Number: Q7SZF1) is the zebrafish homolog of the UPF0489 protein C5orf22 found in humans . This protein belongs to a family of evolutionarily conserved proteins with largely uncharacterized functions. In zebrafish (Danio rerio), zgc:56556 serves as an important model for studying the conserved functions of this protein family across vertebrate species.

The significance of studying this protein in zebrafish stems from several research advantages:

• Zebrafish embryos develop externally and are transparent, allowing for easy visualization of developmental processes

• High genetic homology with humans for many conserved proteins

• Well-established genetic manipulation techniques for functional studies

• Ability to rapidly generate large numbers of offspring for statistical analyses

Researchers investigating zgc:56556 typically examine its expression patterns during development, potential roles in specific tissues, and functional implications through knock-down or knock-out studies.

What are the validated applications for zgc:56556 Antibody?

The zgc:56556 Antibody (CSB-PA763067XA01DIL) has been validated for the following applications :

This antibody has been specifically verified to produce positive results with recombinant immunogen protein/peptide . When designing experiments, researchers should note that while these applications have been verified by the manufacturer, optimization for specific experimental conditions may be required.

How should zgc:56556 Antibody be stored and handled for optimal performance?

Proper storage and handling of zgc:56556 Antibody is critical for maintaining its performance over time:

Storage conditions:

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

• Avoid repeated freeze-thaw cycles as this can lead to denaturation and loss of antibody activity

• The antibody is supplied in a buffer containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative

Handling recommendations:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• When removing from storage, thaw on ice

• Centrifuge briefly before opening the tube to ensure all contents are at the bottom

• Work with antibody solutions on ice when possible

• Return to appropriate storage conditions immediately after use

Proper temperature management is particularly important for polyclonal antibodies to maintain epitope recognition capacity over the shelf-life of the product.

What control samples should be included when using zgc:56556 Antibody?

Rigorous experimental design with appropriate controls is essential for generating reliable results with zgc:56556 Antibody:

Essential controls for zgc:56556 Antibody experiments:

• Positive control: The antibody package includes 200μg of recombinant immunogen protein/peptide that can serve as a positive control

• Negative control: The included 1ml pre-immune serum should be used as a negative control to establish baseline and non-specific binding

• Secondary antibody-only control: Samples treated with only secondary antibody (no primary antibody) to identify non-specific binding of the detection system

• Blocking peptide control: If available, competition with the immunizing peptide can confirm specificity

• Biological negative controls:

  • Wild-type tissue where the protein is not expressed

  • Morpholino knockdown or CRISPR/Cas9 knockout samples to demonstrate antibody specificity

  • Developmental stage-specific controls if the protein has temporal expression patterns

This comprehensive control strategy follows practices similar to those used in other antibody validation studies, such as those for Zika virus antibodies where multiple control types were utilized to ensure specificity .

What are the optimal protocols for Western blot using zgc:56556 Antibody?

When performing Western blot with zgc:56556 Antibody, the following optimized protocol is recommended:

Sample preparation:

• Extract proteins from zebrafish tissues using RIPA buffer supplemented with protease inhibitors

• Determine protein concentration using BCA or Bradford assay

• Load 20-50μg of protein per lane (optimize based on expression level)

SDS-PAGE separation:

• Use 10-12% polyacrylamide gels based on the expected molecular weight

• Include molecular weight markers

Transfer and blocking:

• Transfer to PVDF membrane (0.45μm) at 100V for 1 hour in cold transfer buffer

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

Antibody incubation:

• Dilute zgc:56556 Antibody in blocking buffer (optimization required, starting from 1:1000)

• Incubate overnight at 4°C with gentle rocking

• Wash 3x10 minutes with TBST

Detection:

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour at room temperature

• Wash 3x10 minutes with TBST

• Develop using ECL substrate and image using appropriate detection system

Special considerations:

• Given that this is an affinity-purified polyclonal antibody, some batch-to-batch variation may occur

• The antibody has been validated specifically for recognizing the recombinant protein immunogen

• For challenging samples, consider using the recombinant immunogen protein included with the antibody package as a positive control

This protocol incorporates methodological approaches similar to those used for other polyclonal antibodies described in the literature .

How can zgc:56556 Antibody be adapted for immunohistochemistry in zebrafish tissues?

While zgc:56556 Antibody is not specifically validated for immunohistochemistry (IHC) by the manufacturer, researchers can adapt it for this application using the following protocol:

Tissue preparation:

• Fix embryos or adult tissues in 4% PFA for 24 hours at 4°C

• Dehydrate through an ethanol series and embed in paraffin

• Section at 5-7μm thickness on charged slides

Antigen retrieval (critical step):

• Deparaffinize and rehydrate sections

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

• Allow to cool slowly to room temperature

Staining procedure:

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

• Block non-specific binding with 5% normal goat serum in PBST (1 hour)

• Apply zgc:56556 Antibody at 1:100-1:500 dilution (requires optimization)

• Incubate overnight at 4°C in a humidified chamber

• Wash 3x5 minutes with PBST

• Apply HRP-conjugated secondary antibody and develop with DAB

• Counterstain with hematoxylin, dehydrate, and mount

Validation steps:

• Include sections treated with pre-immune serum as negative controls

• Perform peptide competition controls where available

• Include developmental stages or tissues known to have different expression levels

• Consider dual labeling with other markers to confirm expression patterns

This approach follows similar methodological principles used in immunohistochemical analysis of other zebrafish proteins and is adapted from approaches used with other polyclonal antibodies .

What strategies can address cross-reactivity concerns with zgc:56556 Antibody?

When working with polyclonal antibodies like zgc:56556 Antibody, addressing potential cross-reactivity is crucial for experimental validity:

Cross-reactivity assessment strategies:

• Sequence alignment analysis:

  • Perform in silico analysis comparing the zgc:56556 protein sequence with related zebrafish proteins

  • Identify regions of high homology that might contribute to cross-reactivity

• Immunoprecipitation-Mass Spectrometry:

  • Use the antibody to immunoprecipitate proteins from zebrafish lysates

  • Analyze precipitated proteins by mass spectrometry to identify potential cross-reactive proteins

  • This approach has been successfully used to characterize antibody specificity in other systems

• Knockout/knockdown validation:

  • Compare antibody reactivity in wild-type versus zgc:56556 knockout or knockdown samples

  • True specificity would be demonstrated by loss of signal in the knockout/knockdown samples

• Recombinant protein panel testing:

  • Test antibody reactivity against a panel of recombinant proteins with structural similarity

  • Include both the target protein and potential cross-reactive proteins

• Epitope mapping:

  • Use overlapping peptide arrays to identify the specific epitopes recognized by the antibody

  • Knowledge of epitope regions can inform potential cross-reactivity based on sequence conservation

These strategies have been effectively employed in other antibody characterization studies, such as those for Zika virus antibodies where extensive cross-reactivity testing was necessary to distinguish between related flaviviruses .

How can zgc:56556 Antibody be used in co-immunoprecipitation studies to identify protein interactions?

Co-immunoprecipitation (Co-IP) can reveal physiologically relevant protein interactions. For zgc:56556 Antibody:

Protocol for Co-IP with zgc:56556 Antibody:

• Sample preparation:

  • Harvest zebrafish tissues or cells of interest

  • Lyse in non-denaturing buffer (e.g., 50mM Tris-HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1% Triton X-100 with protease inhibitors)

  • Clear lysate by centrifugation (14,000×g, 10 minutes, 4°C)

• Pre-clearing (reduces non-specific binding):

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5μg of zgc:56556 Antibody to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 50μl Protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with lysis buffer

  • Elute bound proteins with SDS sample buffer

• Analysis of interacting proteins:

  • Separate by SDS-PAGE

  • Analyze by:

    • Western blot for suspected interacting proteins

    • Silver staining followed by mass spectrometry for unbiased identification

• Critical controls:

  • Pre-immune serum IP as negative control

  • Input sample (10% of lysate used for IP)

  • IgG control (non-specific rabbit IgG)

• Validation of interactions:

  • Confirm key interactions by reverse Co-IP

  • Verify physiological relevance through functional assays

  • Consider proximity ligation assay for in situ confirmation

This approach is modeled after co-immunoprecipitation strategies used with other specific antibodies, as described in literature for protein interaction studies .

What are common troubleshooting strategies for weak or non-specific signals with zgc:56556 Antibody?

When encountering issues with zgc:56556 Antibody, systematic troubleshooting can identify and resolve experimental problems:

For weak signals:

• Antibody concentration:

  • Increase antibody concentration incrementally (e.g., from 1:1000 to 1:500, 1:250)

  • Extended incubation times (overnight at 4°C)

• Antigen retrieval (for tissue sections):

  • Test alternative antigen retrieval methods:

    • Citrate buffer (pH 6.0)

    • EDTA buffer (pH 8.0)

    • Enzymatic retrieval with proteinase K

• Protein loading:

  • Increase total protein amount loaded

  • Enrich for the protein of interest through subcellular fractionation

• Detection system:

  • Switch to more sensitive detection systems (e.g., SuperSignal West Femto)

  • Consider tyramide signal amplification for IHC/ICC

For non-specific signals:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time to 2 hours or overnight at 4°C

• Washing stringency:

  • Increase wash buffer stringency (add 0.1-0.3% SDS or 0.5M NaCl)

  • Extend washing times and number of washes

• Antibody dilution:

  • Use higher dilutions to reduce non-specific binding

  • Prepare antibody dilutions in blocking buffer with 0.05% Tween-20

• Pre-absorption:

  • Pre-absorb antibody with non-specific proteins (e.g., acetone powder from non-target tissues)

• Cross-reactivity elimination:

  • Pre-incubate with recombinant proteins of potential cross-reactive targets

This systematic approach to troubleshooting follows practices utilized in antibody optimization studies across various research applications .

How can zgc:56556 Antibody be validated for studies across different developmental stages in zebrafish?

Developmental validation of zgc:56556 Antibody requires methodical approach to account for temporal expression changes:

Validation across developmental stages:

• Expression profiling:

  • Collect zebrafish embryos at key developmental stages: cleavage (0-3 hpf), blastula (3-5 hpf), gastrula (5-10 hpf), segmentation (10-24 hpf), pharyngula (24-48 hpf), and larval (48 hpf-30 dpf)

  • Extract RNA for RT-qPCR to determine transcript expression pattern

  • Extract protein for Western blot using zgc:56556 Antibody

  • Compare protein and transcript levels to ensure antibody detection correlates with gene expression

• Spatial validation by whole-mount immunohistochemistry:

  • Process embryos at different stages for whole-mount immunohistochemistry

  • Optimize fixation conditions for each stage (typically 4% PFA for 2-24 hours depending on stage)

  • Compare antibody staining patterns with in situ hybridization data for the zgc:56556 transcript

  • Document spatiotemporal expression patterns

• Specificity controls specific to developmental studies:

  • Morpholino knockdown at different stages to confirm signal reduction

  • CRISPR/Cas9 knockout embryos as negative controls

  • Rescue experiments with mRNA injection in knockdown embryos

  • Western blot analysis of stage-specific lysates to confirm antibody detects appropriately sized protein

• Application-specific optimization:

  • Adjust permeabilization protocols for different stages (longer treatments for later stages)

  • Modify antigen retrieval methods as appropriate for developmental stage

  • Adjust antibody concentration for different stages based on expression levels

This developmental validation approach follows methodologies similar to those employed in characterizing stage-specific protein expression in zebrafish using other antibodies, as described in literature on developmental protein studies .

How can zgc:56556 Antibody be used for comparative studies across different fish species?

Cross-species applications of zgc:56556 Antibody require careful consideration of evolutionary conservation and epitope preservation:

Cross-species application methodology:

• Sequence homology analysis:

  • Perform sequence alignment of zgc:56556 across target fish species

  • Calculate percent identity and similarity within epitope regions

  • Species with >70% identity in epitope regions are more likely to show cross-reactivity

• Western blot validation in multiple species:

  • Extract proteins from equivalent tissues across different fish species

  • Run side-by-side Western blots with zgc:56556 Antibody

  • Include positive control (zebrafish samples) and negative controls

  • Compare band patterns and molecular weights across species

• Immunohistochemical cross-validation:

  • Perform parallel IHC on tissue sections from different species

  • Use identical protocols initially, then optimize as needed

  • Compare staining patterns with known expression data or RNA localization

  • Include absorption controls with recombinant proteins

• Epitope-specific considerations:

  • For highly conserved regions, maintain standard antibody concentrations

  • For less conserved regions, reduce stringency of washing or increase antibody concentration

  • Consider pre-absorption with non-specific proteins to reduce background

• Documentation and validation standards:

  • Document all cross-reactivity testing comprehensively

  • Validate function-specific interactions through additional methods (e.g., mass spectrometry)

  • Consider raising species-specific antibodies for critical applications

This approach is similar to the cross-species validation performed with other conserved protein antibodies in comparative biology studies .

What methodologies can be applied to investigate zgc:56556 protein function using this antibody?

The zgc:56556 Antibody can be employed in multiple methodological approaches to elucidate protein function:

Functional investigation strategies:

• Subcellular localization studies:

  • Perform immunofluorescence with zgc:56556 Antibody in zebrafish cells or tissue sections

  • Co-stain with markers for cellular compartments (nucleus, ER, Golgi, mitochondria)

  • Use confocal microscopy to determine precise localization

  • Confirm findings with subcellular fractionation and Western blotting

• Protein-protein interaction networks:

  • Use zgc:56556 Antibody for immunoprecipitation followed by mass spectrometry

  • Validate key interactions with co-immunoprecipitation and reverse co-immunoprecipitation

  • Perform proximity ligation assays to confirm interactions in situ

  • Map interaction domains through domain deletion constructs

• Dynamic expression analysis:

  • Monitor protein expression changes in response to developmental cues or environmental stimuli

  • Compare protein levels with transcript levels to identify post-transcriptional regulation

  • Use pulse-chase experiments with metabolic labeling to determine protein turnover rates

• Loss-of-function phenotype correlation:

  • Generate CRISPR/Cas9 knockout or morpholino knockdown zebrafish

  • Analyze phenotypic consequences of zgc:56556 depletion

  • Correlate phenotypes with expression patterns revealed by immunohistochemistry

  • Perform rescue experiments with wild-type or mutant constructs

• Post-translational modification analysis:

  • Use zgc:56556 Antibody to immunoprecipitate the protein from tissues

  • Analyze post-translational modifications by mass spectrometry

  • Generate phospho-specific or other modification-specific antibodies if relevant

These methodological approaches align with strategies used in functional characterization studies of other proteins where specific antibodies played a critical role in elucidating biological function .

How might zgc:56556 Antibody contribute to understanding evolutionary conservation of UPF0489 protein family function?

The zgc:56556 Antibody offers opportunities for investigating evolutionary conservation across vertebrates:

Evolutionary research approaches:

• Comparative expression mapping:

  • Apply zgc:56556 Antibody in zebrafish developmental stages alongside homologs in other vertebrates

  • Map tissue-specific expression patterns across species to identify conserved expression domains

  • Correlate expression with conserved developmental programs

• Structure-function relationships:

  • Use the antibody to immunoprecipitate the protein complex from zebrafish

  • Compare interacting partners with those of homologs in other species

  • Identify evolutionarily conserved protein interactions as indicators of core functional domains

• Functional conservation testing:

  • Express human C5orf22 in zgc:56556 knockout zebrafish

  • Use the antibody to confirm expression and localization of the human protein

  • Assess functional rescue as evidence of conserved function

  • Compare interactomes of native and rescue proteins

• Developmental program involvement:

  • Use the antibody to track protein expression during key evolutionary conserved developmental processes

  • Apply in genetic backgrounds with perturbations to conserved pathways

  • Identify signaling networks that regulate this protein family across species

This evolutionary approach follows methodologies employed in comparative studies of conserved proteins across vertebrate species, providing insights into fundamental biological processes .

What considerations apply when using zgc:56556 Antibody in conjunction with other research tools?

Integrating zgc:56556 Antibody with other research methodologies requires strategic planning:

Multimodal research integration:

• Compatibility with fluorescent proteins:

  • When using zgc:56556 Antibody in tissues expressing fluorescent proteins (e.g., GFP transgenic lines):

    • Select secondary antibodies with non-overlapping emission spectra

    • Confirm that fixation conditions preserve both fluorescent protein signal and epitope recognition

    • Consider using Quantum dots or other distinctive labels for multiplexing

• Integration with CRISPR/Cas9 genome editing:

  • Design experiments to validate antibody specificity using CRISPR knockouts

  • Use the antibody to confirm protein loss in F0 mosaic mutants

  • Apply in phenotypic rescue experiments with modified versions of the protein

• Combination with live imaging approaches:

  • Design experimental timelines where live imaging precedes fixation and immunostaining

  • Establish protocols for relocating specific cells/regions after processing

  • Consider correlative light and electron microscopy approaches

• RNA-based methods integration:

  • Compare protein localization (antibody) with mRNA expression (in situ hybridization)

  • Integrate with RNA-seq data to correlate transcript and protein levels

  • Use with ribosome profiling to investigate translational regulation

• Mass spectrometry complementation:

  • Use antibody for immunoprecipitation prior to mass spectrometry

  • Validate mass spectrometry-identified modifications with immunoblotting

  • Apply for protein complex purification before structural analysis