FKBP53 Antibody

What is FKBP53 and what is its primary function?

FKBP53 is an FK506-binding protein (FKBP) type immunophilin that possesses histone chaperone activity. In Arabidopsis (AtFKBP53), it has been demonstrated to play a crucial role in chromatin remodeling and transcriptional regulation. The protein contains a typical peptidylprolyl isomerase (PPIase) domain and several highly charged domains. Its primary function appears to be repressing ribosomal gene expression, particularly 18S rDNA genes, by acting at the chromatin level . The protein represents one of seven multi-domain FK506-binding proteins identified in some plant species and functions in nucleosome assembly through its interaction with histones, particularly histone H3 .

What are the key structural domains of FKBP53?

FKBP53 is a multidomain protein with a distinctive structure that enables its various functions. The protein contains:

• A typical peptidylprolyl isomerase (PPIase) domain

• Several highly charged domains, particularly charged acidic domains

• N-terminal domain (NTD, residues 1-100/112 in AtFKBP53)

• C-terminal domain (CTD, residues 360-477 in AtFKBP53, containing the FKBD)

Research has demonstrated that while the protein possesses the PPIase domain, this domain is actually dispensable for its histone chaperone activity. Instead, the charged acidic domains are sufficient for histone binding and chaperone activity, highlighting the modular nature of this protein's function .

How do I select the appropriate FKBP53 antibody for my research?

When selecting an FKBP53 antibody for research applications, consider these key factors:

• Species specificity: Determine if the antibody detects FKBP53 from your species of interest. Some antibodies, like those developed for FKBP51, demonstrate cross-reactivity across human, mouse, and rat samples, but specificity for FKBP53 should be verified for your target species .

• Application compatibility: Verify the antibody has been validated for your intended application (Western blotting, immunohistochemistry, immunoprecipitation, etc.). Different antibody clones may perform optimally in different applications.

• Epitope location: For studies examining specific domains or post-translational modifications, select antibodies that target relevant epitopes or modifications.

• Validation data: Review available scientific data showing the antibody's performance in relevant applications, including published literature and manufacturer validation data .

• Positive and negative controls: Plan to include appropriate controls in your experimental design to confirm antibody specificity, including knockout cell lines where available.

How can I distinguish between different phosphorylated isoforms of FKBP53?

Distinguishing between various phosphorylated isoforms of FKBP53 requires specialized techniques and considerations:

• Resolutive electrophoresis: Utilize high-percentage (15%) PAGE/SDS with extended low-voltage (10V) overnight runs to effectively separate phosphorylated isoforms .

• Phospho-specific antibodies: Use antibodies specifically targeting phosphorylated amino acids to detect phosphorylation status. These can be combined with general FKBP53 antibodies in parallel blots or sequential probing to identify specific isoforms .

• Phosphatase treatment validation: Confirm phosphorylation by treating samples with alkaline phosphatase, which should eliminate phosphorylation-dependent bands or shifts as demonstrated with FKBP51 .

• 2D gel electrophoresis: For comprehensive analysis of multiple phosphorylation states, employ 2D electrophoresis (isoelectric focusing followed by SDS-PAGE) to separate proteins by both charge and size.

• Mass spectrometry: For precise identification of phosphorylation sites, use phosphopeptide enrichment followed by mass spectrometry analysis, which can map specific phosphorylated residues within the protein sequence.

Note that research on FKBP51 has shown that stress conditions such as oxidative stress or fasting can influence phosphorylation and cellular localization, potentially translating to similar regulatory mechanisms for FKBP53 .

What experimental approaches can verify FKBP53 interactions with chromatin components?

Verifying FKBP53 interactions with chromatin components requires multiple complementary approaches:

• Chromatin Immunoprecipitation (ChIP): This technique has been successfully used to demonstrate AtFKBP53 association with 18S rDNA gene chromatin. ChIP followed by qPCR or sequencing can identify specific genomic regions where FKBP53 binds .

• Co-immunoprecipitation (Co-IP): This method can confirm physical interactions between FKBP53 and histone proteins or other chromatin modifiers. Studies have shown AtFKBP53 interacts with histone H3 through its acidic domains . For optimal results:

  • Use mild lysis conditions (TEGM buffer: TES pH 7.6, 50 mM NaCl, 4 mM EDTA, 10% glycerol)

  • Include phosphatase inhibitors (1 mM NaF, 10 mM Na₂O₄V)

  • Add protease inhibitors to preserve protein integrity

• Nucleosome assembly assays: These have been instrumental in demonstrating the histone chaperone activity of AtFKBP53. The assays can distinguish which domains are responsible for specific functions, such as the finding that charged acidic domains are sufficient for histone chaperone activity .

• Fluorescence microscopy/co-localization: Immunolocalization with confocal microscopy has been used to visualize subcellular localization and confirm interactions. For instance, FKBP53 has been shown to co-localize with HDT1 in the nucleolus .

• Yeast Two-Hybrid (Y2H) assays: This approach has successfully identified interactions between FKBP53 and other proteins, such as HDT1 and effector proteins from pathogens .

How can I design experiments to investigate FKBP53's role in transcriptional regulation?

To investigate FKBP53's role in transcriptional regulation, consider these experimental approaches:

• Gene expression analysis in knockout/knockdown models:

  • Generate FKBP53 knockout or knockdown lines using CRISPR-Cas9 or RNAi

  • Perform RNA-seq or targeted qRT-PCR to identify genes affected by FKBP53 deletion

  • Research has shown that 18S rDNA genes are overexpressed when AtFKBP53 activity is reduced or eliminated

• ChIP-seq analysis:

  • Perform ChIP-seq to map genome-wide binding sites of FKBP53

  • Correlate binding sites with transcriptional changes in knockout/knockdown models

  • Analyze histone modification patterns at FKBP53-bound regions

• Reporter gene assays:

  • Design reporter constructs containing promoters of interest fused to reporter genes (GUS, luciferase)

  • Compare reporter activity in wild-type vs. FKBP53-deficient backgrounds

  • Analyze effects of FKBP53 overexpression on reporter activity

• Domain function analysis:

  • Create constructs expressing mutant versions of FKBP53 lacking specific domains

  • Assess which domains are necessary for transcriptional repression activity

  • Previous research has shown that the PPIase domain is dispensable for histone chaperone activity, while acidic domains are sufficient

• Stress response experiments:

  • Expose cells to various stressors (oxidative stress, nutrient deprivation)

  • Monitor changes in FKBP53 phosphorylation, localization, and target gene expression

  • Similar approaches with FKBP51 have revealed stress-induced translocation from mitochondria to nucleus

What are the optimal conditions for FKBP53 antibody validation?

Proper validation of FKBP53 antibodies requires systematic approaches to ensure specificity and reliability:

• Western blot optimization:

  • For standard detection: 9% PAGE/SDS run at 75V for approximately 1 hour

  • For resolving phosphorylated isoforms: 15% PAGE/SDS run at 10V overnight in a cold room

  • Transfer to PVDF membranes followed by blocking with appropriate buffer

  • Test cross-reactivity with related FKBP family members (FKBP12, FKBP13, FKBP38, FKBP52, etc.)

• Specificity controls:

  • Compare signal between wild-type and FKBP53 knockout cell lines/tissues

  • Preabsorption tests with recombinant FKBP53 protein

  • Parallel testing with multiple antibodies targeting different epitopes

  • Use siRNA knockdown samples as negative controls

• Cross-species reactivity assessment:

  • Test antibody against FKBP53 from different species if working with non-human models

  • Verify epitope conservation across species through sequence alignment

• Application-specific validation:

  • For immunohistochemistry: test on positive control tissues with known FKBP53 expression

  • For ChIP applications: verify enrichment of known target loci (e.g., 18S rDNA genes)

  • For immunofluorescence: confirm subcellular localization patterns match literature reports

What are the best practices for preparing recombinant FKBP53 for antibody production?

When preparing recombinant FKBP53 for antibody production or experimental use, follow these best practices:

• Expression construct design:

  • Use codon-optimized ORF for expression in E. coli

  • Consider different tag options (His-tag, GST-tag) depending on downstream applications

  • For antibody production, full-length protein may generate antibodies with broader applications

  • For domain-specific studies, create constructs for individual domains (NTD, CTD/FKBD)

• Expression conditions:

  • Use E. coli BL21(DE3) cells grown in 2× YT media with 100 μg/ml ampicillin

  • Induce expression with 0.2 mM IPTG at 18°C for 16 hours when culture reaches OD600 of 0.5

  • These conditions have been optimized for AtFKBP53 expression

• Protein purification:

  • For His-tagged proteins:

    • Resuspend cells in buffer containing 50 mM Tris (pH 7.5), 500 mM NaCl, 1 mM β-mercaptoethanol, 10 mM imidazole, and 1 mM PMSF

    • Lyse cells using ultrasonic processing

    • Clarify lysate by centrifugation

    • Purify using nickel affinity chromatography

    • Elute with buffer containing 20 mM Tris (pH 7.5), 300 mM NaCl, 1 mM β-mercaptoethanol, 1 mM PMSF, and 500 mM imidazole

  • For GST-tagged proteins:

    • Use similar lysis conditions but purify using glutathione-based affinity chromatography

• Quality control:

  • Verify protein identity using mass spectrometry

  • Assess purity by SDS-PAGE

  • Confirm structural integrity through circular dichroism or activity assays

  • Test for endotoxin contamination if the protein will be used for immunization

How should FKBP53 antibodies be stored and handled for optimal performance?

For optimal performance of FKBP53 antibodies, follow these storage and handling recommendations:

• Long-term storage:

  • Store at -20°C to -70°C for up to 12 months from date of receipt

  • Use a manual defrost freezer to avoid temperature fluctuations

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Working stock preparation:

  • After reconstitution, store at 2-8°C under sterile conditions for up to 1 month

  • For longer storage of reconstituted antibody, aliquot and freeze at -20°C to -70°C for up to 6 months

  • Label aliquots with date of reconstitution and dilution information

• Dilution optimization:

  • Optimal dilutions should be determined empirically for each application

  • Generally, starting dilutions:

    • Western blot: 0.3-1.0 μg/mL

    • Immunohistochemistry: 5-15 μg/mL

    • Immunoprecipitation: 2-4 μg per sample

• Buffer considerations:

  • For Western blotting, specific buffer systems may influence results

  • Immunoblot Buffer Group 1 has been used successfully for detecting FKBP proteins

  • When detecting phosphorylated isoforms, include phosphatase inhibitors in all buffers

• Secondary antibody selection:

  • Choose appropriate species-specific secondary antibodies

  • If using rat-derived primary antibodies, HRP-conjugated Anti-Rat IgG

  • For goat-derived antibodies, HRP-conjugated Anti-Goat IgG

How can I address non-specific binding in FKBP53 antibody applications?

Non-specific binding can complicate interpretation of results when using FKBP53 antibodies. Here are methodological approaches to address this issue:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, milk proteins, commercial blockers)

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Include 0.1-0.3% Tween-20 in washing and antibody dilution buffers

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Higher dilutions may reduce background while maintaining specific signal

  • Western blot experiments with FKBP proteins have successfully used 0.3-1.0 μg/mL concentrations

• Cross-reactivity assessment:

  • Test against recombinant FKBP family members (FKBP12, FKBP13, FKBP38, FKBP52)

  • Some FKBP antibodies show less than 1% cross-reactivity with related family members

  • Include knockout or knockdown controls to confirm specificity

• Detection system considerations:

  • If using chemiluminescence, reduce substrate incubation time

  • Consider fluorescent secondary antibodies for more quantitative results

  • Use specialized detection systems like Simple Western™ which has been validated for FKBP proteins

• Sample preparation improvements:

  • Include additional washing steps between incubations

  • Pre-clear lysates with protein A/G beads before immunoprecipitation

  • Use reducing conditions for Western blot applications

What factors affect the detection of FKBP53 in different subcellular compartments?

FKBP53 localization can change under different conditions, affecting detection in subcellular compartments:

• Stress-induced relocalization:

  • Oxidative stress and fasting can induce translocation of related FKBP proteins from mitochondria to nucleus

  • Such relocalization may affect antibody accessibility and detection efficiency

  • Consider fixation methods that preserve native protein distribution

• Fixation protocol optimization:

  • For immunofluorescence/immunohistochemistry:

    • Paraformaldehyde (4%) works well for general protein detection

    • Methanol fixation may better preserve nuclear proteins

    • For detecting nucleolar proteins like FKBP53, immersion fixed paraffin-embedded sections have been successful

• Nuclear extraction methods:

  • When analyzing nuclear FKBP53:

    • Use specialized nuclear extraction buffers

    • Include phosphatase inhibitors to preserve phosphorylation status

    • Gentle homogenization methods to prevent nuclear damage

• Co-localization considerations:

  • FKBP53 co-localizes with HDT1 in the nucleolus

  • Use confocal microscopy with appropriate nuclear and nucleolar markers

  • Z-stack imaging to confirm true co-localization versus overlapping signals

• Post-translational modification effects:

  • Phosphorylation status may affect antibody recognition

  • Different antibodies may have varying sensitivities to phosphorylated isoforms

  • Consider using phosphatase treatment as a control when analyzing subcellular distribution

How can I optimize ChIP protocols for studying FKBP53 interactions with chromatin?

Optimizing ChIP protocols for FKBP53 requires attention to several methodological details:

• Chromatin preparation:

  • Optimize crosslinking conditions (1% formaldehyde for 10-15 minutes is standard)

  • Test different sonication parameters to achieve DNA fragments of 200-500 bp

  • Verify sonication efficiency by agarose gel electrophoresis

  • Include protease and phosphatase inhibitors in all buffers

• Antibody selection and validation:

  • Test multiple antibodies targeting different epitopes of FKBP53

  • Perform preliminary ChIP-qPCR on known targets (18S rDNA genes) to validate antibody performance

  • Include IgG control and input samples for normalization

  • Consider ChIP-grade antibodies or validate standard antibodies for ChIP applications

• Washing conditions optimization:

  • Adjust stringency of wash buffers based on signal-to-noise ratio

  • Increase number of washes to reduce background

  • Consider including detergents like SDS or Triton X-100 at appropriate concentrations

• Target loci selection:

  • Include known FKBP53 binding sites as positive controls (rRNA genes)

  • Design primers for both promoter and gene body regions

  • Include negative control regions (genes not regulated by FKBP53)

• Data analysis considerations:

  • Normalize to input DNA

  • Compare enrichment to IgG control

  • For genome-wide studies, include spike-in controls for quantitative comparisons

  • Correlate binding data with expression data to establish functional relationships

What are the best methods for studying FKBP53's histone chaperone activity?

To study FKBP53's histone chaperone activity, the following methodological approaches are recommended:

• Nucleosome assembly assays:

  • In vitro nucleosome reconstitution using purified histones and DNA

  • Monitor nucleosome formation by gel mobility shift assays

  • Compare wild-type FKBP53 with domain deletion mutants to identify functional domains

  • These assays have demonstrated that AtFKBP53's charged acidic domains are sufficient for histone chaperone activity

• Histone binding assays:

  • Pull-down assays using tagged FKBP53 and purified histones

  • Co-immunoprecipitation from cell lysates

  • Quantitative binding assays such as isothermal titration calorimetry (ITC)

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Research has shown AtFKBP53 interacts with histone H3 through its acidic domains

• Domain function analysis:

  • Create constructs expressing:

    • Full-length FKBP53

    • N-terminal domain only (NTD)

    • C-terminal domain only (CTD/FKBD)

    • Constructs lacking specific charged domains

  • Express and purify these constructs as described previously

  • Compare their histone binding and nucleosome assembly activities

• Chromatin structure analysis:

  • Micrococcal nuclease (MNase) digestion assays to assess nucleosome positioning

  • Compare chromatin structure in wild-type vs. FKBP53-deficient cells

  • Focus on regions known to be regulated by FKBP53, such as rRNA genes

• In vivo functional assays:

  • Monitor transcriptional changes of target genes in response to FKBP53 manipulation

  • Assess chromatin accessibility using techniques like ATAC-seq

  • Analyze histone modification patterns at FKBP53-regulated loci

How can immunoprecipitation techniques be optimized for studying FKBP53 protein interactions?

To optimize immunoprecipitation techniques for studying FKBP53 protein interactions:

• Buffer optimization:

  • Use TEGM buffer (TES pH 7.6, 50 mM NaCl, 4 mM EDTA, 10% glycerol, 20 mM Na₂O₄Mo)

  • Supplement with phosphatase inhibitors (1 mM NaF, 10 mM Na₂O₄V)

  • Include protease inhibitor mixture

  • These conditions have been successful for immunoprecipitating FKBP proteins

• Antibody selection and amount:

  • For tagged FKBP53 (e.g., FLAG-tagged), use 2 μL anti-FLAG mouse IgG (clone M2)

  • For endogenous FKBP53, use 2-5 μg of validated anti-FKBP53 antibody

  • Always include non-immune IgG as negative control

  • Rotate samples for 2.5 hours at 4°C for optimal binding

• Protein capture methods:

  • Use 15 μL protein A-Sepharose for antibody capture

  • For antibodies with lower affinity to protein A, use protein G beads

  • Consider magnetic beads for reduced background

  • Wash pellets three times with 1 mL of buffer to reduce non-specific binding

• Elution and detection strategies:

  • For Western blot detection:

    • Resolve proteins on 9% PAGE/SDS (75V, ~1 h) for standard running

    • Use 15% PAGE/SDS (10 V, overnight) for resolving phosphorylated isoforms

    • Transfer to Immobilon-P membranes

    • Develop with ECL detection systems

• Verification of interactions:

  • Validate interactions using alternate approaches (Y2H, proximity ligation assay)

  • Perform reciprocal IPs (IP with antibody to interacting protein, detect FKBP53)

  • Use cells expressing tagged versions of both proteins for confirmation

  • For known interactions, such as FKBP53 with HDT1, compare to published results

What emerging techniques could enhance our understanding of FKBP53 function and regulation?

Several emerging techniques hold promise for advancing our understanding of FKBP53:

• Proximity labeling approaches:

  • BioID or TurboID fusion proteins to identify proximal interacting partners

  • APEX2-based proximity labeling for identifying transient interactions

  • These methods could reveal novel FKBP53 interaction partners in different cellular compartments

• Single-cell approaches:

  • Single-cell RNA-seq to identify cell-specific effects of FKBP53

  • Single-cell ATAC-seq to examine chromatin accessibility changes

  • These techniques could reveal heterogeneity in FKBP53 function across cell populations

• Live-cell imaging technologies:

  • FRAP (Fluorescence Recovery After Photobleaching) to study dynamics of FKBP53-chromatin interactions

  • FRET-based sensors to monitor FKBP53 conformational changes or protein interactions

  • Optogenetic approaches to control FKBP53 activity with spatial and temporal precision

• Cryo-EM and structural biology:

  • High-resolution structural analysis of FKBP53 in complex with histones

  • Structural studies of FKBP53 bound to chromatin

  • These approaches could provide mechanistic insights into FKBP53's histone chaperone activity

• CRISPR-based epigenome editing:

  • Targeted recruitment of FKBP53 to specific genomic loci

  • CUT&RUN or CUT&Tag for high-resolution mapping of FKBP53 binding sites

  • These techniques could help establish causal relationships between FKBP53 binding and transcriptional outcomes

How might FKBP53 antibodies contribute to understanding plant-pathogen interactions?

FKBP53 antibodies can provide valuable insights into plant-pathogen interactions:

• Monitoring FKBP53 during infection:

  • Track changes in FKBP53 expression, localization, and modification during pathogen infection

  • Compare FKBP53 dynamics in resistant versus susceptible plant varieties

  • Research has shown that FKBP53 interacts with effector proteins from pathogens like Heterodera schachtii

• Studying effector-triggered immunity:

  • Investigate how pathogen effectors like 32E03 interact with FKBP53

  • Analyze changes in FKBP53-mediated gene regulation during infection

  • Examine co-localization of FKBP53 with pathogen effectors in the nucleolus

• Chromatin immunoprecipitation during infection:

  • Map changes in FKBP53 binding to chromatin during pathogen challenge

  • Identify genes whose regulation by FKBP53 is altered during infection

  • Correlate these changes with defense gene expression

• Protein complex analysis:

  • Identify changes in FKBP53-containing protein complexes during infection

  • The known interaction between FKBP53 and HDT1 might be affected by pathogen effectors

  • Immunoprecipitation with FKBP53 antibodies followed by mass spectrometry could reveal infection-specific interactions

• Transgenic approaches:

  • Use FKBP53 antibodies to validate knockout or overexpression lines

  • Monitor FKBP53 levels in plants expressing pathogen effectors

  • These approaches could help establish the significance of FKBP53 in plant immunity