FTL Recombinant Monoclonal Antibody

What is FTL and what biological functions does it serve?

FTL (Ferritin Light Chain) is a crucial subunit of the ferritin protein complex that stores iron in a soluble, non-toxic, readily available form. It plays a fundamental role in iron homeostasis within cells. The FTL protein is involved in several key biological processes, including:

• Iron uptake in the ferrous form and deposition as ferric hydroxides after oxidation

• Delivery of iron to cells throughout the body

• Mediation of iron uptake in capsule cells of the developing kidney

• Delivery to lysosomes via the cargo receptor NCOA4 for autophagic degradation and release of iron

These functions make FTL an important research target for studies involving iron metabolism, oxidative stress responses, and related pathological conditions.

What applications are FTL recombinant monoclonal antibodies validated for?

FTL recombinant monoclonal antibodies have been validated for multiple research applications, with specific validation depending on the manufacturer and antibody clone. Common validated applications include:

• IHC (Immunohistochemistry) on formalin-fixed paraffin-embedded (FFPE) tissues

• WB (Western Blot) for protein detection and quantification

• ELISA (Enzyme-Linked Immunosorbent Assay) for protein quantification

• FC (Flow Cytometry) for cellular analysis

• Protein Array applications for high-throughput screening

When selecting an antibody for your specific research needs, it's important to verify that the antibody has been validated for your intended application and target species.

What is the difference between mouse monoclonal and recombinant monoclonal FTL antibodies?

The key differences between mouse monoclonal and recombinant monoclonal FTL antibodies involve their production methods, consistency, and application advantages:

Mouse Monoclonal FTL Antibodies:

• Produced using traditional hybridoma technology

• Generated by immunizing mice with the target antigen (FTL protein or fragment)

• Antibody-producing B cells from the mouse are fused with myeloma cells to create hybridomas

• Each hybridoma cell line produces a single antibody specificity

• Examples include mouse monoclonal antibody FTL/1386 (ab218400)

Recombinant Monoclonal FTL Antibodies:

• Produced using in vitro cloning and recombinant DNA technology

• The antibody genes are incorporated into expression vectors and introduced into host cells

• Expression occurs in a cell culture environment

• Purified from the supernatant of transfected host cells through affinity chromatography

• Can be produced in different host species (e.g., rabbit IgG)

Recombinant antibodies typically offer greater batch-to-batch consistency, reduced animal use, and can be engineered for enhanced performance in specific applications.

How should I optimize immunohistochemistry protocols when using FTL recombinant antibodies?

Optimizing immunohistochemistry protocols with FTL recombinant antibodies requires careful consideration of several factors:

Antigen Retrieval:
For FFPE tissues, heat-induced epitope retrieval is typically necessary. Based on validated protocols:

• Boil tissue sections in 10mM Citrate Buffer, pH 6.0, for 10-20 minutes

• Allow sections to cool at room temperature for 20 minutes before proceeding

Antibody Dilution:

• Start with the manufacturer's recommended dilution range (typically 1:50-1:200 for IHC)

• Perform a dilution series to determine optimal antibody concentration

• For ab218400, successful staining has been reported at 0.2-0.5 μg/ml

Incubation Conditions:

• Optimal incubation time appears to be 30 minutes to overnight at 4°C

• Room temperature incubation (30 minutes) has been validated for some antibodies

Detection Systems:

• Select an appropriate detection system based on your species and isotype (e.g., HRP-polymer for rabbit IgG)

• Include appropriate blocking steps to minimize background

Controls:

• Always include positive tissue controls (human pancreas and testis tissues have shown positive staining)

• Include negative controls (primary antibody omission and isotype controls)

• Consider using FTL knockout cell lines as specificity controls when available

What are the critical considerations for using FTL antibodies in Western blot applications?

When using FTL antibodies for Western blot applications, researchers should consider these methodological aspects:

Sample Preparation:

• Use appropriate lysis buffers that preserve protein structure

• Include protease inhibitors to prevent degradation

• Determine optimal protein loading (20 μg per lane has been validated)

Running Conditions:

• Use reducing conditions for optimal results with FTL antibodies

• Ensure appropriate percentage gel selection (FTL has a predicted band size of 20 kDa)

Antibody Concentration:

• Use at dilutions between 1:500-1:1000 for optimal results

• Higher concentrations may increase background signal

Expected Results:

• The predicted band size for FTL is 20 kDa

• Some antibodies may also detect additional bands at 31 kDa and 35 kDa

• Validate specificity using knockout cell lines when possible

Detection Systems:

• Both chemiluminescence and fluorescent detection systems have been validated

• For fluorescent detection, secondary antibodies such as IRDye® 800CW and IRDye® 680RD have been successfully used

What controls should be included when validating FTL antibody specificity?

Proper controls are essential for validating antibody specificity in FTL-related research:

Positive Controls:

• Well-characterized cell lines with known FTL expression (HeLa, A549, HepG2)

• Human tissue samples known to express FTL (liver, pancreas, testis)

• Recombinant FTL protein for western blot positive controls

Negative Controls:

• FTL knockout cell lines (several are commercially available):

  • FTL knockout HeLa cell line (ab265534)

  • FTL knockout HAP1 cell line

• Primary antibody omission controls

• Isotype controls matching the primary antibody's species and isotype

Loading Controls:

• For western blots, include housekeeping proteins like:

  • GAPDH (ab181602)

  • Alpha-tubulin (ab52866)

Peptide Competition:

• Consider performing peptide competition assays using the immunizing peptide to confirm binding specificity

The most stringent specificity validation comes from comparing wild-type to knockout samples, as demonstrated in the published validation data showing antibody reactivity in wild-type HeLa cells but signal loss in FTL knockout HeLa cells .

How can FTL recombinant antibodies be utilized in studying iron metabolism disorders?

FTL recombinant antibodies can be powerful tools for investigating iron metabolism disorders through several advanced research approaches:

Tissue Expression Analysis:

• IHC analysis of patient tissues can reveal altered FTL expression patterns in hereditary hyperferritinemia-cataract syndrome, neuroferritinopathy, and other iron storage disorders

• Comparative analysis between healthy and diseased tissues can identify cell-specific changes in FTL expression

Protein-Protein Interaction Studies:

• Co-immunoprecipitation using FTL antibodies can identify altered interactions between FTL and:

  • Ferritin heavy chain (FTH)

  • NCOA4 (Nuclear Receptor Coactivator 4), which mediates ferritinophagy

  • Other iron regulatory proteins

Subcellular Localization Studies:

• Immunofluorescence with FTL antibodies can reveal changes in ferritin localization in disease states

• Co-localization with lysosomal markers can assess ferritinophagy efficiency

Quantitative Analysis:

• Western blotting and ELISA with FTL antibodies allow quantitative assessment of FTL expression levels

• Flow cytometry can be used to assess cellular FTL content on a single-cell level

Genetic Modification Models:

• FTL knockout cell lines can serve as models for studying the functional consequences of FTL deficiency

• Complementation studies with wild-type or mutant FTL can assess functional impacts of specific mutations

These approaches enable comprehensive investigation of molecular mechanisms underlying iron metabolism disorders and potential therapeutic interventions.

What methodological considerations are important when studying FTL in autophagy and ferritinophagy?

Studying FTL in autophagy and ferritinophagy requires specific methodological considerations:

Induction of Ferritinophagy:

• Iron chelation with deferoxamine (DFO) or other chelators to induce ferritinophagy

• Starvation conditions to trigger general autophagy

• Use of specific autophagy inducers like rapamycin

Dual Immunostaining Approach:

• Co-staining for FTL and NCOA4 (the selective cargo receptor for ferritinophagy)

• Co-staining for FTL and autophagy markers (LC3, p62)

• Co-staining for FTL and lysosomal markers (LAMP1, LAMP2)

Live-Cell Imaging Considerations:

• Transfection with fluorescently tagged FTL constructs

• Use of lysosomal dyes in combination with immunofluorescence

• Time-lapse imaging to capture dynamic ferritinophagy processes

Quantitative Assessments:

• Western blot analysis of FTL degradation kinetics under ferritinophagy-inducing conditions

• Flow cytometry to quantify cellular FTL levels during ferritinophagy

• ELISA-based quantification of FTL in cellular compartments

Inhibitor Studies:

• Use of autophagy inhibitors (bafilomycin A1, chloroquine) to block lysosomal degradation

• Assessment of FTL accumulation when lysosomal function is inhibited

• Comparison between general autophagy inhibition and specific ferritinophagy inhibition

Knockout/Knockdown Verification:

• NCOA4 knockout/knockdown to confirm specificity of ferritinophagy process

• FTL knockout cells as negative controls

• Autophagy-related gene knockouts to determine pathway dependencies

These methodological approaches allow for comprehensive characterization of FTL's role in ferritinophagy and the broader autophagy pathway.

How can FTL recombinant antibodies be used in multiplexed imaging applications?

Multiplexed imaging with FTL recombinant antibodies enables simultaneous visualization of multiple targets, providing spatial context for understanding FTL's role in cellular processes:

Sequential Multiplexing Approach:

• Use FTL antibodies in combination with antibodies to other proteins of interest

• Ensure antibodies are from different host species or isotypes to prevent cross-reactivity

• Consider using specifically validated antibody panels for iron metabolism studies

Spectral Unmixing Strategies:

• Use fluorophores with minimal spectral overlap

• Employ spectral imaging systems capable of separating closely overlapping fluorophores

• Validated fluorophore combinations include IRDye® 800CW and IRDye® 680RD

Multi-epitope Ligand Cartography (MELC):

• Use FTL antibodies in iterative immunofluorescence imaging cycles

• Photobleach fluorophores between cycles to build high-parameter tissue maps

• Combine with image analysis tools for quantitative spatial analysis

Mass Cytometry Imaging:

• Conjugate FTL antibodies to rare earth metals for mass cytometry imaging (IMC)

• Allows for highly multiplexed imaging (30+ markers) with minimal signal overlap

• Particularly useful for complex tissue microenvironments

Proximity Ligation Assays:

• Combine FTL antibodies with antibodies against potential interaction partners

• Detect protein-protein interactions within intact cells or tissues

• Particularly useful for studying FTL interactions with FTH or NCOA4

Validation Controls:

• Include single-stain controls for each antibody

• Use FTL knockout samples as negative controls

• Include fluorescence minus one (FMO) controls for accurate gating and analysis

These multiplexed approaches significantly enhance our understanding of FTL's spatial relationships with other cellular components in both normal and pathological conditions.

What are common causes of false positive or false negative results when using FTL antibodies?

Understanding potential sources of error is crucial for accurate interpretation of FTL antibody-based experiments:

Common Causes of False Positives:

Common Causes of False Negatives:

Researchers should systematically evaluate these factors when troubleshooting unexpected results, giving particular attention to proper controls and validated protocols.

How can I quantitatively analyze FTL expression data from different experimental platforms?

Quantitative analysis of FTL expression requires platform-specific approaches and careful normalization:

Western Blot Quantification:

• Use digital imaging systems for densitometric analysis

• Normalize FTL band intensity to loading controls (GAPDH, alpha-tubulin)

• Construct standard curves using recombinant FTL protein for absolute quantification

• Use FTL knockout samples to determine background signal threshold

Immunohistochemistry Quantification:

• Employ digital pathology software for automated scoring

• Quantify parameters such as:

  • Percentage of positive cells

  • Staining intensity (0, 1+, 2+, 3+)

  • H-score calculation (% of 1+ cells × 1) + (% of 2+ cells × 2) + (% of 3+ cells × 3)

• Compare to validated positive controls (human pancreas, testis)

Flow Cytometry Analysis:

• Gate populations based on forward/side scatter properties

• Establish negative gates using FTL knockout cells or isotype controls

• Quantify median fluorescence intensity (MFI) for population analysis

• Consider fluorescence minus one (FMO) controls for precise gating

ELISA Data Analysis:

• Generate standard curves using purified recombinant FTL

• Ensure samples fall within the linear range of the standard curve

• Account for matrix effects through sample dilution series

• Calculate intra- and inter-assay coefficients of variation for quality control

Cross-Platform Data Integration:

• Normalize data to common reference samples across platforms

• Use relative quantification when comparing between methods

• Consider statistical approaches like z-score normalization for multi-platform comparisons

• Validate findings using orthogonal methods

These quantitative approaches enhance experimental rigor and reproducibility in FTL expression studies.

How should I resolve contradictory results between different detection methods for FTL?

When facing contradictory results across different detection methods for FTL, a systematic approach to resolution is necessary:

Methodological Analysis:

• Evaluate each method's sensitivity limits and dynamic range

• Consider epitope accessibility differences between techniques

• Assess whether the antibodies used recognize different epitopes

• Review whether post-translational modifications affect epitope recognition

Validation Hierarchy:
Consider results from multiple methods with this hierarchy of confidence:

• Methods using knockout controls (highest confidence)

• Methods using multiple antibodies recognizing different epitopes

• Methods with comprehensive positive and negative controls

• Methods without adequate controls (lowest confidence)

Sample-Specific Considerations:

• Different subcellular localization may affect detection efficiency

• Sample preparation differences may alter epitope exposure

• Protein complexes may mask epitopes in certain assays

• Isoform-specific expression may cause apparent contradictions

Resolution Strategies:

Documentation Practices:

• Record detailed methodological parameters for each experiment

• Document lot numbers of antibodies used

• Maintain comprehensive records of all controls

• Consider pre-registering experimental protocols to minimize bias

How can FTL recombinant antibodies be utilized in studying neurodegenerative disorders?

FTL recombinant antibodies offer valuable tools for investigating the role of iron dysregulation in neurodegenerative conditions:

Neuroferritinopathy Studies:

• IHC analysis of brain tissues to detect abnormal FTL accumulation

• Co-staining with markers of neurodegeneration to establish spatial relationships

• Quantitative analysis of FTL expression in different brain regions

• Detection of mutant FTL forms associated with specific disorders

Iron-Related Neurodegeneration:

• Assessment of FTL expression in Parkinson's, Alzheimer's, and ALS models

• Correlation of FTL levels with markers of oxidative stress

• Investigation of FTL's role in neuronal iron homeostasis

• Monitoring changes in FTL expression during disease progression

Methodological Considerations:

• Optimize tissue fixation to preserve brain tissue architecture

• Use antigen retrieval optimized for neural tissues (citrate buffer, pH 6.0)

• Consider autofluorescence quenching for aged brain tissues

• Include age-matched controls for comparative analysis

Cellular Models:

• Use of patient-derived iPSCs differentiated into neurons

• FTL knockout neuronal models for functional studies

• Transfection with wild-type or mutant FTL to study functional consequences

• Live-cell imaging to track FTL dynamics in neuronal cells

These approaches enable investigation of iron dysregulation as both a contributor to and consequence of neurodegenerative processes, potentially revealing new therapeutic targets.

What considerations are important when developing multiplex assays incorporating FTL antibodies?

Developing effective multiplex assays with FTL antibodies requires careful attention to several technical aspects:

Antibody Compatibility:

• Select antibodies from different host species or isotypes

• Verify lack of cross-reactivity between antibodies

• Test for epitope competition when using multiple antibodies against the same protein

• Validate each antibody individually before combining into multiplex format

Signal Optimization:

• Titrate each antibody to determine optimal working concentration

• Balance signal intensities across all targets

• Consider sequential detection for targets with vastly different abundance

• Optimize detection reagents for each antibody in the panel

Panel Design for Iron Biology:
Effective multiplex panels might include:

Technical Validation:

• Perform single-stain controls for each antibody

• Include fluorescence minus one (FMO) controls

• Validate with positive controls (human liver, pancreas)

• Use knockout samples as negative controls when available

Data Analysis Considerations:

• Account for spectral overlap through compensation or unmixing

• Develop standardized gating strategies for flow cytometry

• Establish quantitative metrics for image-based multiplexing

• Apply appropriate statistical methods for multiparameter data

These considerations help ensure robust and reliable results when incorporating FTL antibodies into multiplex experimental platforms.

What emerging technologies might enhance the utility of FTL recombinant antibodies in research?

Several cutting-edge technologies are poised to expand the research applications of FTL recombinant antibodies:

Single-Cell Proteomics:

• Integration of FTL antibodies into single-cell mass cytometry (CyTOF)

• Analysis of FTL expression heterogeneity at single-cell resolution

• Correlation with other iron metabolism proteins at single-cell level

• Development of FTL-targeted antibody-oligonucleotide conjugates for CITE-seq

Advanced Imaging Techniques:

• Super-resolution microscopy for nanoscale localization of FTL

• Expansion microscopy for enhanced visualization of FTL distribution

• Correlative light and electron microscopy to relate FTL localization to ultrastructure

• Light sheet microscopy for 3D visualization of FTL in whole tissues or organoids

Engineered Antibody Variants:

• Site-specific conjugation methods for better preservation of antibody function

• Nanobody or single-chain variable fragment (scFv) derivatives for improved tissue penetration

• Bispecific antibodies targeting FTL and interaction partners simultaneously

• Intrabodies for live-cell tracking of FTL dynamics

Artificial Intelligence Applications:

• Machine learning algorithms for automated analysis of FTL staining patterns

• Deep learning approaches for multiplexed image analysis

• Predictive modeling of FTL expression based on multiparameter data

• Computer vision for high-throughput screening applications

These emerging technologies will provide unprecedented insights into FTL biology and iron homeostasis, potentially leading to novel therapeutic strategies for iron-related disorders.

What standardization efforts are needed to improve reproducibility in FTL antibody-based research?

Improving reproducibility in FTL antibody-based research requires systematic standardization efforts:

Antibody Validation Standards:

• Adoption of minimum validation requirements (knockout controls, multiple applications)

• Standardized reporting of validation data in publications

• Independent verification of antibody specificity by third parties

• Creation of publicly accessible validation datasets

Protocol Standardization:

• Development of consensus protocols for common applications

• Standardized fixation and antigen retrieval methods for tissues

• Uniform sample preparation procedures for cell lines

• Standardized reporting formats for experimental conditions

Reference Materials:

• Creation of reference cell lines with defined FTL expression levels

• Development of standard recombinant FTL protein preparations

• Generation of standard tissue microarrays for IHC validation

• Establishment of digital reference images for staining patterns

Data Reporting Requirements:

• Minimum information standards for FTL antibody experiments

• Mandatory reporting of antibody catalog numbers and lot numbers

• Comprehensive documentation of controls used

• Full disclosure of image acquisition and processing parameters

Community Resources:

• Central database of validated FTL antibodies and applications

• Repository of optimized protocols for different experimental contexts

• Collaborative quality assessment programs

• Cross-laboratory validation initiatives

These standardization efforts would significantly enhance data reproducibility and comparability across different laboratories, accelerating progress in FTL-related research fields.