zgc:66022 Antibody

What is zgc:66022 and what functional roles does it play in cellular processes?

zgc:66022 is the zebrafish gene symbol for the Mitochondrial Fission Factor (MFF), a protein involved in several critical cellular processes . Functionally, MFF plays a significant role in both mitochondrial and peroxisomal fission . It facilitates the recruitment and association of dynamin-related protein 1 (DNM1L) to the mitochondrial surface, which is essential for the mitochondrial fission process . Additionally, MFF may be involved in the regulation of synaptic vesicle membrane dynamics through its ability to recruit DNM1L to clathrin-containing vesicles .

The gene has several aliases across different species, including mffa, C2orf33, and GL004, with UniProt identifiers including Q7SZQ4 (zebrafish) and Q9GZY8 (human) .

What types of zgc:66022/MFF antibodies are currently available for research?

Research laboratories currently have access to both polyclonal and monoclonal antibodies targeting zgc:66022/MFF:

These antibodies have been validated for different applications and offer varying degrees of species cross-reactivity, with some specific to zebrafish and others cross-reacting with human, mouse, and/or rat orthologs .

How should zgc:66022 antibodies be stored for optimal stability and performance?

Storage conditions vary by antibody formulation, but following these guidelines will help maintain antibody integrity:

• Short-term storage (weeks): Store at 4°C for monoclonal antibodies like C2orf33 Recombinant Rabbit Monoclonal Antibody (HL1312)

• Long-term storage (months to years): Store at -20°C, specifically for polyclonal antibodies

• Some formulations contain stabilizers: PBS with 50% glycerol and 0.02% sodium azide (pH 7.4)

• Avoid repeated freeze-thaw cycles which can lead to antibody degradation

• Centrifuge briefly prior to opening the vial to collect solution at the bottom

• Store as concentrated solution for maximum stability

When retrieving an antibody from long-term storage, allow it to equilibrate to room temperature gradually before opening to prevent condensation, which can introduce contamination and accelerate degradation.

What applications are zgc:66022 antibodies validated for?

Current zgc:66022/MFF antibodies have been validated for multiple research applications:

The methodological approach should be optimized based on the specific antibody used. For example, when using antibodies for immunohistochemistry, optimal dilutions should be determined empirically, as the required concentration can vary based on tissue type, fixation method, and detection system used.

How can specificity and sensitivity of zgc:66022 antibodies be validated in experimental models?

Validating antibody specificity is crucial for reliable research outcomes. A comprehensive validation protocol includes:

• Expression Verification:

  • Compare protein expression in tissues/cells known to express high levels of MFF (e.g., Jurkat cells) versus those with low expression

  • Include positive controls such as recombinant MFF protein

• Knockdown/Knockout Validation:

  • Test antibody on samples from MFF-knockout models or cells treated with MFF-targeting siRNA

  • A valid antibody will show reduced or eliminated signal in these samples

• Multiple Detection Methods:

  • Confirm findings using at least two different detection techniques (e.g., WB and IHC)

  • For example, combine Western blotting with immunohistochemistry as demonstrated in research for other proteins such as ZNF32

• Peptide Competition Assay:

  • Pre-incubate the antibody with its immunizing peptide before application

  • A specific antibody's signal should be blocked in the presence of excess peptide

• Cross-Reactivity Assessment:

  • Test against known homologs in different species (zebrafish, human, mouse)

  • Analyze sequence similarity between the immunizing peptide and potential cross-reactive proteins

A rigorous validation approach similar to that used for mAb-pZNF32-8D9 could be employed, where the clone positive to the peptide showed 92% positivity in ELISA testing .

What methodologies are effective for studying MFF-DNM1L interactions using zgc:66022 antibodies?

MFF's interaction with dynamin-related protein 1 (DNM1L) is critical for mitochondrial fission. These methodological approaches can be employed:

• Co-Immunoprecipitation (Co-IP):

  • Use anti-zgc:66022 antibody to precipitate MFF and associated proteins

  • Western blot the precipitate with anti-DNM1L antibodies

  • Include appropriate controls: IgG control, input sample, and reverse Co-IP

• Proximity Ligation Assay (PLA):

  • Utilize zgc:66022 antibodies in combination with DNM1L antibodies from different host species

  • Quantify fluorescent spots indicating protein-protein interaction within 40 nm distance

  • Analyze subcellular localization of interactions

• FRET (Fluorescence Resonance Energy Transfer):

  • Label zgc:66022 antibody and DNM1L antibody with compatible fluorophores

  • Measure energy transfer as indicator of close proximity

  • Control for spectral overlap and direct excitation of acceptor

• Immunofluorescence Co-localization:

  • Use zgc:66022 antibodies alongside DNM1L antibodies in fixed cells

  • Quantify co-localization using Pearson's or Mander's coefficients

  • Include mitochondrial markers (e.g., MitoTracker) to confirm localization at mitochondria

• Live Cell Imaging:

  • Use fluorescently tagged Fab fragments derived from zgc:66022 antibodies

  • Monitor recruitment dynamics in real-time

  • Correlate with mitochondrial fission events

When selecting antibodies for these applications, consider using conformational epitope-recognizing antibodies like 4D06 rather than those recognizing linear epitopes, as they may more effectively capture native protein interactions .

How can zgc:66022 antibodies be optimized for quantitative studies of mitochondrial fission?

Quantitative assessment of mitochondrial fission using zgc:66022 antibodies requires rigorous methodological approaches:

• Standardized Immunofluorescence Protocol:

  • Optimize fixation methods to preserve mitochondrial morphology

  • Use consistent antibody concentrations and incubation times

  • Include calibration standards in each experiment for normalization

• High-Content Imaging Analysis:

  • Develop automated image acquisition and analysis pipelines

  • Measure parameters such as MFF puncta per mitochondrial surface area

  • Incorporate machine learning algorithms for pattern recognition

• Quantitative Western Blotting:

  • Use purified recombinant MFF protein to generate standard curves

  • Implement digital image analysis with appropriate software

  • Include housekeeping protein controls and mitochondrial markers for normalization

• Flow Cytometry Approach:

  • Develop protocols for intracellular staining of MFF

  • Combine with mitochondrial dyes to analyze correlation

  • Set up multiparameter analysis to correlate MFF levels with mitochondrial mass

• ELISA-Based Quantification:

  • Develop sandwich ELISA using capture and detection antibodies

  • Generate standard curves using recombinant MFF protein

  • Optimize sample preparation to ensure consistent protein extraction

For accurate quantification, incorporate methodological approaches similar to those used for developing high-sensitivity detection systems as demonstrated in the ZNF32 antibody development case, where ELISA positivity reached 92% sensitivity .

What considerations are important when using zgc:66022 antibodies across different species models?

Cross-species applications require careful consideration of sequence homology and epitope conservation:

• Epitope Mapping and Conservation Analysis:

  • Compare amino acid sequences of zgc:66022/MFF across target species

  • Focus on antibodies targeting highly conserved regions for cross-species applications

  • For example, analyze the conservation of the region corresponding to residues F129-S179 of human MFF that some antibodies target

• Validation in Each Species Model:

  • Perform Western blots on tissue samples from each species

  • Include positive controls (tissues known to express high levels of MFF)

  • Document any differences in banding patterns or molecular weights

• Species-Specific Optimization:

  • Adjust antibody concentrations for each species

  • Modify incubation times and washing conditions as needed

  • Consider species-specific secondary antibodies to reduce background

• Documentation of Cross-Reactivity:

  • Create a comprehensive table of validated species reactivity:

• Alternative Approaches for Non-Compatible Species:

  • Consider developing species-specific antibodies when cross-reactivity is poor

  • Use antibodies against conserved post-translational modifications

  • Implement epitope tagging strategies in model organisms

Leveraging approaches similar to broadly neutralizing antibody development could help identify conserved epitopes that function across species barriers .

What are common issues when using zgc:66022 antibodies and how can they be resolved?

Researchers frequently encounter these challenges when working with zgc:66022/MFF antibodies:

• High Background in Immunostaining:

  • Increase blocking time and concentration (use 5-10% serum from the species of secondary antibody)

  • Add 0.1-0.3% Triton X-100 for better antibody penetration

  • Implement longer and more vigorous washing steps

  • Use more dilute antibody concentrations after titration tests

• Multiple Bands in Western Blots:

  • Verify if bands represent different MFF isoforms or splice variants

  • Include peptide competition controls to identify specific bands

  • Optimize lysis conditions to reduce protein degradation

  • Use freshly prepared samples and include protease inhibitors

• Weak or No Signal:

  • Test different epitope retrieval methods for fixed tissues

  • Increase antibody concentration or incubation time

  • Ensure target protein is not degraded during sample preparation

  • Verify expression of zgc:66022/MFF in your sample type

• Inconsistent Results Between Experiments:

  • Standardize all experimental conditions (fixation, blocking, antibody dilutions)

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Include positive control samples in each experiment

  • Document and control for lot-to-lot variations

• Non-specific Binding:

  • Pre-absorb antibody with tissue/cell lysates from species of interest

  • Use more stringent washing conditions (higher salt concentration)

  • Test monoclonal alternatives if polyclonal antibodies show non-specificity

  • Consider using antibodies purified by antigen affinity chromatography

How can zgc:66022 antibodies be effectively used in combination with other mitochondrial markers?

Multiplexed detection of MFF with other mitochondrial proteins requires:

• Antibody Compatibility Planning:

  • Select primary antibodies from different host species

  • Use isotype-specific secondary antibodies to prevent cross-reactivity

  • Verify spectral separation of fluorophores to minimize bleed-through

• Sequential Staining Protocol:

  • For same-species antibodies, use sequential rather than simultaneous incubation

  • Block with Fab fragments between staining steps

  • Validate each antibody individually before combining

• Recommended Marker Combinations:

  • MFF + DNM1L: To study recruitment of fission machinery

  • MFF + TOM20: To distinguish outer membrane localization

  • MFF + DRP1: To analyze phosphorylation-dependent interactions

  • MFF + mitochondrial matrix markers: To correlate fission events with matrix segregation

• Controls for Co-localization Studies:

  • Include single-stained samples to set compensation

  • Use co-localization standards

  • Perform pixel shift analysis as negative control

• Quantitative Co-localization Analysis:

  • Employ appropriate software (ImageJ with JACoP, Imaris, etc.)

  • Use appropriate statistical measures (Pearson's, Mander's coefficients)

  • Analyze sufficient number of cells for statistical significance

These approaches can be integrated with lessons from other successful antibody development programs, such as those used for viral antibody research, emphasizing careful validation at each step .

What approaches can maximize specificity when studying zgc:66022 in complex tissue samples?

Complex tissues present unique challenges for specific detection of zgc:66022/MFF:

• Optimized Antigen Retrieval:

  • Test multiple methods (heat-induced vs. enzymatic)

  • Optimize pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval time for different tissue types

• Tissue-Specific Blocking Strategies:

  • Use tissue-matched normal serum (5-10%)

  • Add 0.1-0.3% Triton X-100 for membrane permeabilization

  • Include specific blockers for endogenous biotin, peroxidase, or phosphatase

• Signal Amplification Methods:

  • Implement tyramide signal amplification for low-abundance targets

  • Use polymer-based detection systems

  • Consider biotin-streptavidin amplification with proper controls

• Background Reduction Techniques:

  • Pre-absorb antibodies against tissue homogenates

  • Use shorter incubation times at higher antibody concentrations

  • Implement avidin-biotin blocking for tissues with high biotin content

• Validation Controls:

  • Include zgc:66022/MFF knockout or knockdown tissues as negative controls

  • Use tissues known to have high MFF expression as positive controls

  • Perform antibody dilution series to determine optimal concentration

• Cell Type-Specific Analysis:

  • Combine with cell type-specific markers in multiplexed immunofluorescence

  • Use laser capture microdissection for subsequent biochemical analysis

  • Consider in situ hybridization for mRNA as complementary approach

Similar methodological rigor to that used in developing therapeutic antibodies would ensure high specificity in complex samples .

How might zgc:66022 antibodies be utilized in studying mitochondrial dynamics in disease models?

zgc:66022/MFF antibodies offer significant potential for investigating mitochondrial dynamics in various disease contexts:

• Neurodegenerative Disease Models:

  • Analyze MFF expression and localization in Alzheimer's, Parkinson's, and ALS models

  • Correlate MFF levels with mitochondrial fragmentation and neuronal death

  • Develop high-throughput screening assays using zgc:66022 antibodies to identify compounds that normalize mitochondrial dynamics

• Cancer Research Applications:

  • Compare MFF expression between normal and tumor tissues using immunohistochemistry

  • Investigate correlation between MFF upregulation and metabolic reprogramming in cancer cells

  • Develop targeted therapies based on cancer-specific alterations in MFF expression

• Cardiovascular Disease Research:

  • Study MFF-mediated fission in cardiomyocytes during ischemia-reperfusion injury

  • Analyze the relationship between mitochondrial fragmentation and cardiac dysfunction

  • Test therapeutic approaches targeting MFF to protect against heart failure

• Mitochondrial Disease Models:

  • Characterize compensatory changes in MFF expression in primary mitochondrial disorders

  • Develop patient-derived cellular models and analyze MFF localization

  • Screen for compounds that modify MFF activity to normalize mitochondrial network

• Metabolic Disorder Research:

  • Investigate MFF regulation in insulin resistance and diabetes

  • Analyze MFF-mediated adaptations to different metabolic states

  • Develop interventions targeting MFF to improve metabolic flexibility

This research could build upon methodologies developed for therapeutic antibodies, adapting them to create tools that can both detect and potentially modulate MFF function in disease contexts .

What innovations in antibody engineering could improve zgc:66022 antibody functionality for advanced applications?

Several cutting-edge approaches could enhance zgc:66022 antibody functionality:

• Affinity Maturation Techniques:

  • Implement computational modeling combined with experimental library screening as demonstrated for the F5 antibody

  • Apply Rosetta-based and dTERMen informatics approaches to improve binding affinity

  • Develop phage display libraries and screen for variants with enhanced binding properties

• Humanization for Therapeutic Development:

  • Convert zgc:66022 antibodies into humanized versions for potential therapeutic applications

  • Apply CDR grafting and framework optimization techniques

  • Implement similar approaches to those used for broadly neutralizing antibodies against viral targets

• Bispecific Antibody Development:

  • Create bispecific antibodies targeting both MFF and interacting proteins (e.g., DNM1L)

  • Design antibodies that can simultaneously bind to MFF and mitochondrial markers

  • Develop bispecific formats that can modulate MFF activity while monitoring its localization

• Intrabody Adaptations:

  • Engineer zgc:66022 antibody fragments for intracellular expression

  • Develop formats stable in reducing cytoplasmic environment

  • Create conditional expression systems for temporal control of intrabody function

• Nanobody and Single-Chain Derivatives:

  • Develop single-domain antibodies against zgc:66022 for improved tissue penetration

  • Create smaller formats for super-resolution microscopy applications

  • Design nanobodies that specifically recognize different conformational states of MFF

Combining these approaches with computational modeling methods as demonstrated in the F5 antibody enhancement study could yield significantly improved reagents .

How can high-throughput screening with zgc:66022 antibodies accelerate mitochondrial biology research?

Implementing high-throughput approaches with zgc:66022 antibodies can revolutionize mitochondrial research:

• Automated Imaging Platforms:

  • Develop high-content screening assays using zgc:66022 antibodies

  • Quantify MFF puncta formation, redistribution, and co-localization

  • Implement machine learning algorithms for pattern recognition and phenotypic classification

• CRISPR-Based Genetic Screens:

  • Use zgc:66022 antibodies to detect phenotypic changes in genome-wide screens

  • Identify novel regulators of MFF localization and function

  • Combine with live-cell imaging for temporal analysis of mitochondrial dynamics

• Drug Discovery Applications:

  • Screen compound libraries for molecules that modulate MFF-dependent fission

  • Develop ELISA-based assays for high-throughput quantification of MFF modifications

  • Create biosensor systems using zgc:66022 antibody fragments for real-time monitoring

• Proteomics Integration:

  • Combine immunoprecipitation using zgc:66022 antibodies with mass spectrometry

  • Identify novel MFF-interacting proteins across different physiological conditions

  • Map post-translational modification landscapes affecting MFF function

• Microfluidic Applications:

  • Develop chip-based systems for rapid antibody-based detection of MFF

  • Create patient sample screening platforms for personalized medicine approaches

  • Implement droplet-based single-cell analysis for heterogeneity studies

These high-throughput approaches could benefit from similar experimental library screening methodologies used in affinity maturation studies for therapeutic antibodies .