IFFO1 Antibody

What is IFFO1 and where is it expressed?

IFFO1 is a protein that belongs to the intermediate filament family, which functions as primordial components of the cytoskeleton and nuclear envelope. It is primarily distributed in the nucleus, followed by the cytoskeleton and cytoplasm. IFFO1 is encoded by the IFFO1 gene in humans and has a calculated molecular weight of 61.98 kDa, although it is commonly observed at 75-85 kDa in Western blots due to post-translational modifications .

The protein contains a highly conserved filament domain spanning 299 amino acids from residue 230 to 529, which has been identified as part of the pfam00038 conserved protein domain family . IFFO1 expression has been detected in various tissues, with notable expression in human and mouse brain and mouse testis tissues .

Functionally, IFFO1 serves as a nuclear matrix protein involved in DNA double-strand break (DSB) repair, specifically in immobilizing broken DNA ends and suppressing chromosome translocation. It interacts with nuclear lamina component LMNA and DNA repair protein XRCC4, forming a nucleoskeleton that relocates to DSB sites .

What applications are IFFO1 antibodies validated for?

IFFO1 antibodies have been validated for multiple research applications with specific sample types:

When selecting an IFFO1 antibody, ensure it has been validated for your specific application and target species. Different antibodies may target different epitopes, which can affect their performance in certain applications. For example, some commercial antibodies target the region spanning amino acids 387-437 .

What are the recommended dilutions for IFFO1 antibody applications?

Optimal dilutions for IFFO1 antibodies vary by application. Based on available data, here are the recommended ranges:

It's important to note that these ranges serve as starting points, and researchers should optimize conditions for their specific experimental system. The optimal dilution may vary depending on factors such as antibody lot, sample type, and detection method . Titration experiments are recommended to determine the ideal antibody concentration that provides maximum specific signal with minimal background.

How should IFFO1 antibodies be stored and handled?

Proper storage and handling are crucial for maintaining antibody activity and specificity. For IFFO1 antibodies, the following conditions are typically recommended:

When working with IFFO1 antibodies:

• Allow the antibody to equilibrate to room temperature before opening the vial

• Briefly centrifuge before use to collect all liquid at the bottom of the tube

• Use clean pipette tips to prevent contamination

• Return to -20°C storage promptly after use

• Handle with appropriate safety precautions as antibodies may contain preservatives like sodium azide

What is the specificity of IFFO1 antibodies?

The specificity of IFFO1 antibodies refers to their ability to recognize IFFO1 protein exclusively without cross-reactivity to other proteins. Commercial IFFO1 antibodies have demonstrated reactivity with human, mouse, and rat samples .

Most IFFO1 antibodies are purified through antigen affinity purification to enhance specificity . For example, one commercial polyclonal antibody is developed using a synthesized peptide derived from human IFFO1 at the amino acid range 387-437 .

When assessing antibody specificity, consider:

• The observed molecular weight in Western blots (75-85 kDa for IFFO1, higher than the calculated 62 kDa due to post-translational modifications)

• Subcellular localization pattern (primarily nuclear with some cytoskeletal/cytoplasmic distribution)

• Validation data from manufacturers, including positive control tissues (brain, testis)

• Cross-reactivity information with related proteins

For critical experiments, additional validation steps such as using IFFO1 knockout cells as negative controls (similar to approaches used with other proteins ) or peptide competition assays may be warranted.

How can IFFO1 antibodies be used to study DNA repair mechanisms?

IFFO1 plays a significant role in DNA repair processes, particularly in immobilizing broken DNA ends and suppressing chromosome translocation during DNA double-strand breaks (DSBs) . IFFO1 antibodies can be valuable tools for investigating these repair mechanisms through several methodological approaches:

Immunofluorescence for localization studies:

• Induce DNA damage in cultured cells using ionizing radiation, radiomimetic drugs, or topoisomerase inhibitors

• Fix cells at various time points after damage induction (15 min to 24 hours)

• Perform immunofluorescence using IFFO1 antibodies (1:10-1:100 dilution)

• Co-stain with markers of DNA damage (γH2AX) and repair proteins (XRCC4, LMNA)

• Analyze colocalization and recruitment kinetics to damage sites

Proximity Ligation Assay (PLA) for protein interactions:

• Use PLA technique to detect in situ interactions between IFFO1 and its known partners XRCC4 and LMNA

• Quantify PLA signals at different time points after DNA damage

• Compare wild-type versus DNA repair-deficient cells to assess functional significance

Chromatin Immunoprecipitation (ChIP) for recruitment to damage sites:

• Induce DNA damage at specific genomic loci using targeted endonucleases

• Perform ChIP using IFFO1 antibodies

• Analyze IFFO1 enrichment at break sites using qPCR or sequencing

• Assess how IFFO1 recruitment correlates with repair outcomes

Biochemical fractionation to study damage-induced relocalization:

• Separate cellular fractions (cytoplasmic, nucleoplasmic, chromatin-bound)

• Analyze IFFO1 distribution before and after DNA damage

• Determine how damage affects IFFO1's association with different cellular compartments

What are the known post-translational modifications of IFFO1?

IFFO1 undergoes extensive post-translational modifications (PTMs) that may affect its function, localization, and interactions with other proteins. Understanding these modifications is crucial for interpreting experimental results with IFFO1 antibodies.

Of particular note is the phosphorylation site at Ser533, which has been identified with high confidence as a specific target for protein kinase C . This phosphorylation might be functionally significant for IFFO1's role in DNA repair.

When working with IFFO1 antibodies, consider that:

• PTMs may affect epitope recognition by antibodies

• The observed molecular weight (75-85 kDa vs. calculated 62 kDa) indicates substantial PTMs

• Different antibodies may have varying sensitivities to modified forms

• PTM status may change dynamically during cellular processes like DNA damage response

For studying specific IFFO1 modifications, phospho-specific antibodies or general PTM detection after IFFO1 immunoprecipitation may be required.

How can I optimize co-immunoprecipitation using IFFO1 antibodies?

Co-immunoprecipitation (Co-IP) is valuable for studying IFFO1's protein interactions, particularly with XRCC4 and LMNA. Here's an optimized protocol for IFFO1 Co-IP experiments:

Buffer preparation:

• Lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with freshly added protease and phosphatase inhibitors

• Wash buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40

• Elution buffer: 0.1 M glycine (pH 2.5)

• Neutralization buffer: 1 M Tris-HCl (pH 8.0)

Co-IP protocol for nuclear proteins like IFFO1:

• Harvest cells and prepare nuclear extracts (important for nuclear matrix proteins)

• Pre-clear lysate with protein A/G beads for 1 hour at 4°C

• Add 2-5 μg of IFFO1 antibody to 500 μg of nuclear extract

• Incubate overnight at 4°C with gentle rotation

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

• Wash beads 4-5 times with wash buffer

• Elute proteins with SDS sample buffer or acid elution

• Analyze by Western blot for IFFO1 and potential interacting proteins

Optimization strategies:

• For studying DNA repair interactions, consider inducing DNA damage before Co-IP

• Include negative controls (IgG from same species as IFFO1 antibody)

• Perform reverse Co-IP (immunoprecipitate XRCC4 or LMNA and probe for IFFO1)

• Cross-link proteins before lysis to stabilize transient interactions

• Adjust salt concentration in wash buffers to optimize specificity vs. sensitivity

Validation approaches:

• Confirm successful IP by probing blot for IFFO1

• Use IFFO1 knockout cells as negative controls

• Include positive controls (known interacting proteins)

• Consider mass spectrometry analysis to identify novel interacting partners

This methodology can be particularly valuable for investigating how IFFO1 functions as a scaffold that allows DNA repair protein XRCC4 and nuclear lamina component LMNA to assemble into a complex at DNA double-strand break sites .

How can I troubleshoot non-specific binding with IFFO1 antibodies?

Non-specific binding is a common challenge when working with antibodies. Here's a systematic approach to troubleshooting non-specific binding with IFFO1 antibodies:

Western blot troubleshooting:

Immunostaining troubleshooting:

General optimization strategies:

• Antibody titration: Test a range of dilutions beyond the recommended range

• Blocking optimization: Test different blocking agents and times

• Washing optimization: Increase number and duration of washes

• Sample preparation refinement: For nuclear proteins like IFFO1, consider nuclear extraction protocols

• Alternative validation: Test a different IFFO1 antibody targeting a different epitope

By systematically working through these troubleshooting steps, researchers can optimize IFFO1 antibody performance and minimize non-specific binding in their experiments.

How can IFFO1 antibodies be used to study interactions with XRCC4 and LMNA?

IFFO1 interacts with XRCC4 (a DNA repair protein) and LMNA (a nuclear lamina component) as part of its role in DNA double-strand break repair . Here are methodological approaches to study these interactions using IFFO1 antibodies:

Proximity Ligation Assay (PLA):

• Fix cells on coverslips before or after DNA damage induction

• Incubate with primary antibodies: anti-IFFO1 + anti-XRCC4 or anti-IFFO1 + anti-LMNA

• Follow standard PLA protocol with appropriate PLA probes

• Visualize interaction foci by fluorescence microscopy

• Quantify signal intensity and number of foci per cell

• Compare patterns before and after DNA damage

This approach allows visualization of protein interactions in situ with high sensitivity and specificity, similar to techniques used in other studies of protein interactions .

Immunofluorescence co-localization:

• Perform triple immunofluorescence staining for IFFO1, XRCC4, and LMNA

• Use confocal microscopy for high-resolution imaging

• Analyze co-localization using quantitative metrics (Pearson's correlation)

• Track changes in co-localization following DNA damage

• Consider super-resolution microscopy for nanoscale interaction details

Biochemical fractionation:

• Separate cellular components into cytoplasmic, nucleoplasmic, and nuclear matrix fractions

• Analyze distribution of IFFO1, XRCC4, and LMNA by Western blot

• Identify fractions containing protein complexes

• Compare patterns before and after DNA damage

Functional validation:

• Generate IFFO1 mutants lacking specific domains

• Express in IFFO1-depleted cells

• Test interaction with XRCC4 and LMNA by co-immunoprecipitation

• Assess functional consequences in DNA repair assays

These complementary approaches can provide insights into how IFFO1 functions as a scaffold that allows XRCC4 and LMNA to assemble into a complex at DNA double-strand break sites, thereby promoting the immobilization of broken ends and preventing chromosome translocation .

What controls should be included when working with IFFO1 antibodies?

Proper controls are essential for generating reliable and interpretable results with IFFO1 antibodies. Here's a comprehensive guide to controls for different experimental applications:

Western blot controls:

• Positive tissue controls: Include mouse brain, human brain, or mouse testis samples where IFFO1 expression has been confirmed

• Molecular weight marker: To verify the observed 75-85 kDa band for IFFO1

• Loading control: Use housekeeping proteins appropriate for your sample type and subcellular fraction

• Negative control: If available, IFFO1 knockout or knockdown samples

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

Immunohistochemistry/Immunofluorescence controls:

• Positive tissue control: Human brain sections where IFFO1 expression is well-documented

• Negative control: Omit primary antibody but include all other steps

• Isotype control: Use non-specific IgG from the same species as the IFFO1 antibody

• Subcellular marker co-staining: Nuclear markers to confirm the predominantly nuclear localization of IFFO1

• Knockdown validation: If available, IFFO1-depleted samples as specificity controls

Immunoprecipitation controls:

• Input sample: Total lysate before immunoprecipitation

• IgG control: Non-specific IgG from the same species as the IFFO1 antibody

• Known interactor positive control: Co-IP of established partners like XRCC4 or LMNA

• Negative control sample: If available, IFFO1 knockout cells or tissues

• Reverse IP: Immunoprecipitate with XRCC4 or LMNA antibodies and probe for IFFO1

General experimental controls:

• Antibody titration: Multiple antibody dilutions to determine optimal concentration

• Validation across applications: Confirm IFFO1 detection using multiple methods

• Cross-species validation: If working with non-human samples, verify antibody reactivity

• Functional controls: For DNA damage studies, include time points before and after damage induction

Implementing these controls will enhance data quality and interpretability when working with IFFO1 antibodies across different experimental applications.

What are the considerations for using IFFO1 antibodies in different tissue types?

When using IFFO1 antibodies across different tissue types, several important considerations should be taken into account:

Tissue-specific expression patterns:
IFFO1 has been detected in human and mouse brain and mouse testis tissues , but expression levels may vary significantly between different tissues and cell types. Higher antibody concentrations or longer incubation times may be needed for tissues with lower IFFO1 expression.

Antigen retrieval optimization:
Different tissues may require modified antigen retrieval protocols:

• For human brain tissue, TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

• Optimization of retrieval conditions (temperature, duration, buffer composition) may be necessary for each tissue type

• For formalin-fixed tissues, more stringent antigen retrieval may be required

Tissue-specific fixation considerations:

• Fixation protocols should be optimized for each tissue type

• Overfixation can mask epitopes and reduce antibody binding

• For neural tissues, shorter fixation times may improve IFFO1 detection

• For frozen sections, brief post-fixation (10 minutes in 4% PFA) may be optimal

Background reduction strategies:

• Autofluorescence varies by tissue (particularly high in brain, liver)

• For brain tissues, Sudan Black B treatment may reduce lipofuscin autofluorescence

• Tissue-specific blocking solutions may be required (e.g., adding serum matching the host species of secondary antibody)

• Increase washing steps for tissues with high background

Antibody penetration:

• Thicker tissue sections may require longer antibody incubation times

• For brain tissue, up to 48-hour antibody incubation at 4°C may improve staining

• Consider using tissue clearing techniques for thick sections

Validation and controls:

• Include known positive tissue controls (brain or testis)

• Use tissue-specific negative controls

• Consider implementing alternative detection methods for confirmation

By carefully optimizing protocols for each tissue type and including appropriate controls, researchers can generate reliable and reproducible results with IFFO1 antibodies across different experimental systems.

How can IFFO1 antibodies be used in ChIP-seq experiments?

While the search results don't specifically mention ChIP-seq experiments with IFFO1 antibodies, methodological guidance can be provided based on IFFO1's known characteristics as a nuclear protein involved in DNA repair:

Antibody selection for ChIP-seq:

• Choose antibodies validated for immunoprecipitation applications

• Polyclonal antibodies often perform better in ChIP than monoclonals

• Consider antibodies targeting regions not involved in DNA binding

• The antibody targeting amino acids 387-437 region may be suitable if validated for IP

ChIP-seq protocol optimization:

• Crosslinking: For nuclear matrix proteins like IFFO1, dual crosslinking with DSG followed by formaldehyde may improve results

• Chromatin preparation: Standard sonication to 200-500 bp fragments

• Immunoprecipitation: Use 3-5 μg of IFFO1 antibody per ChIP reaction

• Controls: Include IgG negative control and input samples

• Library preparation: Follow standard ChIP-seq library protocols

• Sequencing depth: Aim for >30 million reads for good coverage

Experimental designs for IFFO1 ChIP-seq:

• Baseline genomic distribution: Map IFFO1 binding sites under normal conditions

• DNA damage response: Compare binding before and after DNA damage induction

• Co-occupancy studies: Compare with XRCC4, LMNA, and other DNA repair proteins

• Correlation with chromatin features: Integrate with datasets on chromatin accessibility and histone modifications

Data analysis considerations:

• Use peak calling algorithms suitable for transcription factor ChIP-seq

• Consider broader peaks given IFFO1's potential scaffolding function

• Analyze distribution relative to genomic features and nuclear matrix attachment regions

• Compare with DNA damage markers (γH2AX) for functional correlation

Given IFFO1's role in DNA repair and nuclear organization, ChIP-seq could provide valuable insights into its genomic distribution and how it changes in response to DNA damage, potentially revealing mechanisms of chromosome translocation suppression .

What future research directions could benefit from IFFO1 antibodies?

IFFO1 antibodies could enable several promising research directions that build on current understanding of this protein's function:

Mechanistic studies of DNA repair:

• Investigation of how IFFO1 prevents chromosome translocations during DNA repair

• Analysis of IFFO1 recruitment kinetics to different types of DNA damage

• Characterization of the IFFO1-XRCC4-LMNA complex assembly and function

• Exploration of how IFFO1 contributes to the choice between different DNA repair pathways

Nuclear architecture studies:

• Examination of IFFO1's role in nuclear matrix organization

• Investigation of how IFFO1 contributes to 3D genome organization

• Analysis of IFFO1's role in tethering damaged DNA to nuclear substructures

• Characterization of IFFO1's relationship with other nuclear structural proteins

Disease relevance exploration:

• Investigation of IFFO1 alterations in cancers with genomic instability

• Analysis of IFFO1 function in neurodegenerative diseases (given its expression in brain tissue)

• Exploration of IFFO1 variants identified in human population studies

• Examination of IFFO1's role in aging-related DNA damage accumulation

Therapeutic applications:

• Development of approaches to modulate IFFO1 function for cancer therapy

• Exploration of IFFO1 as a biomarker for DNA repair deficiencies

• Investigation of IFFO1 in chemotherapy and radiation resistance mechanisms

Technical innovations:

• Development of phospho-specific IFFO1 antibodies targeting key regulatory sites

• Creation of engineered antibody fragments for live-cell IFFO1 tracking

• Design of conformation-specific antibodies that recognize IFFO1 in different functional states

• Implementation of IFFO1 antibodies in spatial proteomics approaches

These research directions would benefit from continued development and validation of high-quality IFFO1 antibodies targeting different epitopes and suitable for various experimental applications.