RAD54L Antibody

What is RAD54L and why is it important in DNA repair research?

RAD54L (also known as RAD54A, hHR54, hRAD54) is a DNA motor protein with multiple critical roles in homologous recombination (HR) DNA repair. It functions as a 84.4 kilodalton protein that catalyzes branch migration and plays essential roles in repairing DNA double-strand breaks, single-stranded DNA gaps, and stalled or collapsed replication forks .

The significance of RAD54L in research stems from its multifunctional nature:

• It acts as a molecular motor during homology search

• It guides RAD51 ssDNA along donor dsDNA

• It plays essential roles in synaptic complex formation

• It catalyzes both fork regression and restoration of model replication forks

• It restrains replication fork progression in human cells

These functions make RAD54L a crucial target for studying genomic stability, DNA repair mechanisms, and potential therapeutic approaches for cancer, particularly in BRCA1/2-deficient tumors.

What applications are RAD54L antibodies typically used for?

RAD54L antibodies are utilized in multiple research applications to study its expression, localization, and function. Based on available commercial antibodies, the most common applications include:

Most anti-RAD54L antibodies work optimally for human samples, with some showing cross-reactivity with mouse and rat orthologs .

How do I validate the specificity of a RAD54L antibody?

Proper validation of RAD54L antibody specificity is critical to ensure experimental reliability. A comprehensive validation approach should include:

• Positive and negative controls:

  • Positive: Cell lines known to express RAD54L (HeLa, Jurkat, 293 cells)

  • Negative: RAD54L knockout cell lines or RAD54L-depleted cells using siRNA

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide should abolish the specific signal in Western blot

• Multiple detection methods: Confirm specificity using at least two different techniques (e.g., WB and IF)

• siRNA validation: Transfect cells with RAD54L-specific siRNAs and confirm decreased signal compared to control siRNA

• Cross-validation with another antibody: Use multiple antibodies targeting different epitopes of RAD54L

• Molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of RAD54L (approximately 84.4 kDa)

As demonstrated in published research, knockout validation is particularly powerful, as seen in studies using HeLa, Hs578T, and MCF7 RAD54L KO cells compared to parental lines .

How can I optimize RAD54L antibody staining for detecting replication stress?

Optimizing RAD54L antibody staining for replication stress studies requires specific conditions to capture its dynamic localization at stalled replication forks. Based on published protocols:

• Treatment conditions:

  • Induce replication stress with hydroxyurea (3 mM for 2 hours) to enrich RAD54L at replication forks

  • Use low concentrations of replication stress agents (0.1-0.5 mM HU) to study RAD54L's role in fork restraint

• Co-staining recommendations:

  • Include RAD51 as a marker for homologous recombination activity

  • Use PCNA as a control for replication forks (PCNA is downregulated after HU treatment)

  • Consider NUCKS1 as an additional marker, which shows similar enrichment patterns to RAD54L

• Fixation and permeabilization:

  • For immunofluorescence, 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization

  • For improved nuclear staining, consider methanol fixation at -20°C

• Signal amplification strategies:

  • Use labeled secondary antibodies with bright fluorophores

  • Consider tyramide signal amplification for low abundance detection

  • Confocal microscopy with z-stacking improves detection of nuclear foci

For replication stress studies, the iPOND (isolation of proteins on nascent DNA) assay has been shown to effectively capture RAD54L enrichment at stalled replication forks .

What are the critical controls when studying RAD54L-mediated fork reversal using antibodies?

When investigating RAD54L's role in fork reversal, several critical controls are necessary to ensure experimental validity:

• Genetic controls:

  • RAD54L knockout cells as negative controls

  • RAD54L KO cells complemented with wild-type RAD54L for rescue experiments

  • RAD54L KO cells expressing separation-of-function mutations:

    • RAD54L-S49E (deficient in oligomerization)

    • RAD54L-4A (deficient in binding to HJ-like DNA structures)

    • RAD54L-4A/S49E (deficient in both binding to HJs and oligomerization)

    • RAD54L-K189R (ATPase-dead mutant)

• Pathway controls:

  • Include controls for both HLTF/SMARCAL1 and FBH1 pathways of RAD51-mediated fork reversal

  • Use siRNAs targeting HLTF, FBH1, or 53BP1 to distinguish between different fork reversal pathways

• Functional assays:

  • DNA fiber assays to measure replication fork progression

  • S1 nuclease sensitivity assays to detect ssDNA gaps

  • iPOND for analyzing proteins at nascent DNA

  • Analysis of nascent strand degradation in BRCA1/2-deficient backgrounds

• Western blot validation:

  • Confirm knockdown/knockout efficiency

  • Verify expression levels of complemented proteins

  • Monitor levels of interacting proteins (RAD51, BRCA1/2)

The disparate requirements for RAD54L in the two fork reversal pathways (FBH1 vs. HLTF/SMARCAL1) necessitate careful experimental design to distinguish between these roles .

How do expression levels of RAD54L vary across cell lines and how might this affect antibody detection?

RAD54L expression varies significantly across different cell lines, which can impact antibody detection sensitivity and optimal working dilutions. While comprehensive expression data across all cell lines is limited, research indicates:

• High expression cell lines:

  • HeLa cells show robust RAD54L expression and are frequently used for validation

  • Jurkat cells and 293 cells also show good expression levels

  • Cancer cell lines (particularly those with genomic instability) often show upregulated RAD54L expression

• Moderate expression cell lines:

  • MCF7 breast cancer cells

  • Hs578T breast cancer cells

  • These cell lines have been successfully used in RAD54L knockout studies

• Low expression cell lines:

  • RPE-1 (near-normal retinal pigment epithelial cells) may have lower basal expression

  • Primary cells generally have lower expression than immortalized lines

When working with low-expressing cell lines, consider:

• Increasing antibody concentration

• Using more sensitive detection systems

• Longer exposure times for Western blots

• Signal amplification methods for immunofluorescence

• Inducing DNA damage to upregulate RAD54L expression

Expression levels can be further modulated by cell cycle phase (higher in S/G2) and in response to DNA damage agents, which should be considered when designing experiments.

What are the optimal conditions for Western blotting with RAD54L antibodies?

Achieving optimal Western blot results with RAD54L antibodies requires attention to several key parameters:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation status

  • For nuclear protein enrichment, consider nuclear extraction protocols

  • Sonication may improve extraction efficiency

• Gel electrophoresis:

  • Use 8% SDS-PAGE gels due to RAD54L's size (84.4 kDa)

  • Load 20-40 μg total protein per lane

  • Include molecular weight markers spanning 70-100 kDa range

• Transfer conditions:

  • Semi-dry or wet transfer systems (wet transfer often works better for larger proteins)

  • Transfer at lower voltage for longer periods (e.g., 30V overnight at 4°C)

  • Use 0.45 μm pore size PVDF membrane rather than 0.2 μm

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Primary antibody dilutions typically 1:500 to 1:1000

  • Overnight incubation at 4°C generally yields better results than short incubations

  • Secondary antibody incubation for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) is generally sufficient

  • For low abundance samples, consider more sensitive ECL substrates

  • Expected molecular weight: approximately 84.4 kDa

Published protocols have successfully used these conditions to detect RAD54L in various cell lines, with validation through peptide competition and RAD54L knockout cells .

How can I optimize immunofluorescence experiments to detect RAD54L at DNA damage sites?

Detecting RAD54L at DNA damage sites requires specific optimization of immunofluorescence protocols:

• DNA damage induction methods:

  • Ionizing radiation: 2-10 Gy, fix cells 2-6 hours post-irradiation

  • Laser microirradiation: Creates localized DNA damage tracks

  • Chemical agents: Mitomycin C (MMC), hydroxyurea (HU), or Olaparib (PARP inhibitor)

• Fixation and permeabilization:

  • Fix with 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.2% Triton X-100 in PBS (10 minutes)

  • Alternative: methanol fixation (-20°C for 10 minutes)

• Blocking:

  • 5% BSA or normal goat serum in PBS (1 hour at room temperature)

  • Include 0.1% Triton X-100 in blocking buffer to reduce background

• Antibody incubation:

  • Primary antibody dilution: 1:100 to 1:200

  • Incubation: overnight at 4°C or 2 hours at room temperature

  • Secondary antibody: 1:500 dilution, 1 hour at room temperature

  • Include DAPI for nuclear counterstaining

• Co-staining markers:

  • γH2AX: Marker for DNA double-strand breaks

  • RAD51: Co-localizes with RAD54L at homologous recombination sites

  • PCNA or EdU: Replication fork markers

  • BrdU: Marks single-stranded DNA regions under non-denaturing conditions

• Microscopy considerations:

  • Confocal microscopy for better spatial resolution

  • Z-stack imaging to capture nuclear foci in different planes

  • Deconvolution for improved signal-to-noise ratio

For studying RAD54L at stalled replication forks specifically, hydroxyurea treatment (3 mM for 2 hours) has been effective in enriching RAD54L at these sites .

What cross-reactivity issues should I be aware of when using RAD54L antibodies?

Understanding potential cross-reactivity is essential for accurate interpretation of RAD54L antibody results:

• Common cross-reactivity concerns:

  • RAD54B: A paralog of RAD54L with similar structure and function

  • Other SWI2/SNF2 family proteins with helicase domains

  • Proteins with similar molecular weights (80-90 kDa range)

• Species cross-reactivity:

  • Most commercially available antibodies are raised against human RAD54L

  • Mouse and rat reactivity varies by antibody:

    • Some antibodies show good cross-reactivity with mouse RAD54L

    • Others are specific to human RAD54L

  • Sequence homology analysis suggests potential cross-reactivity with canine, porcine, and monkey orthologs, though validation is required

• Epitope considerations:

  • N-terminal targeted antibodies (aa 1-25) : May have different cross-reactivity profiles than C-terminal antibodies

  • Antibodies targeting the conserved ATPase domain may show higher cross-species reactivity

  • Monoclonal antibodies (e.g., [RAD54 4E3/1] or [F-11]) typically offer higher specificity but potentially lower cross-species reactivity

• Validation approaches:

  • Peptide competition assays to confirm specificity

  • Testing in RAD54L knockout cells from multiple species

  • Western blot analysis to confirm the correct molecular weight

  • Performing siRNA knockdown experiments to verify signal reduction

For projects requiring cross-species applications, the Aviva Systems Biology RAD54L antibody targeting the N-terminal region has been reported to react with human, mouse, rabbit, rat, bovine, dog, guinea pig, and zebrafish samples, though independent validation is recommended .

How can RAD54L antibodies be used to study cancer therapy resistance mechanisms?

RAD54L antibodies play a critical role in investigating therapy resistance mechanisms, particularly in relation to DNA repair pathways in cancer:

Research demonstrates that RAD54L functions in two distinct pathways of RAD51-mediated fork reversal, with different requirements in each pathway, making it a valuable target for understanding therapy resistance in BRCA1/2-deficient tumors .

What are the best approaches for studying RAD54L interactions with other DNA repair proteins using antibodies?

Investigating RAD54L interactions with other DNA repair proteins requires specific methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-RAD54L antibodies suitable for IP applications

  • Reverse Co-IP (using antibodies against interaction partners)

  • Nuclear extracts typically yield better results than whole cell lysates

  • Gentle lysis conditions (NP-40 or Triton X-100 rather than RIPA)

  • DNase I treatment to distinguish DNA-mediated from direct protein interactions

• Proximity ligation assay (PLA):

  • Detects protein-protein interactions in situ with <40 nm resolution

  • Requires antibodies from different species for RAD54L and partner proteins

  • Particularly useful for studying interactions at specific cellular structures

• Chromatin immunoprecipitation (ChIP):

  • ChIP-re-ChIP approaches to study co-occupancy at specific genomic loci

  • Combined with DNA damage induction to analyze recruitment kinetics

  • Integration with iPOND technique to study interactions at replication forks

• Key interaction partners to investigate:

  • RAD51: Primary interaction partner in homologous recombination

  • BRCA1/2: Cooperative functions in fork protection and HR

  • FBH1: RAD54L branch migration activity is essential in the FBH1 pathway

  • HLTF/SMARCAL1: RAD54L works downstream of these factors

  • 53BP1: Antagonistic relationship in fork protection

• Functional validation approaches:

  • Separation-of-function mutants (e.g., RAD54L-S49E, RAD54L-4A)

  • Domain mapping through deletion constructs

  • Competitive peptide inhibitors to disrupt specific interactions

Recent research demonstrates disparate requirements for RAD54L in different fork reversal pathways - in the FBH1 pathway, RAD54L's engagement largely depends on its ability to catalyze branch migration, while in the HLTF/SMARCAL1 pathway, RAD54L branch migration activity is dispensable .

How can I develop immunohistochemistry protocols for RAD54L in tissue microarrays and patient samples?

Developing robust immunohistochemistry (IHC) protocols for RAD54L in clinical samples requires careful optimization:

• Tissue preparation and fixation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues: 10% neutral buffered formalin, 24-48 hours

  • Fresh frozen sections: Flash freeze in OCT compound

  • Fixation time is critical - overfixation can mask epitopes

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) with Tris-EDTA buffer (pH 9.0) works well for RAD54L

  • Pressure cooker method: 3 minutes at high pressure

  • Alternative: citrate buffer (pH 6.0) with 20-minute heat treatment

  • Enzymatic retrieval generally less effective for nuclear proteins

• Blocking and antibody parameters:

  • Block with 5% normal serum from secondary antibody host species

  • Additional blocking with avidin/biotin if using ABC detection systems

  • Primary antibody dilution: Start with 1:100-1:200 and optimize

  • Incubation: Overnight at 4°C often yields better results than 1 hour at RT

• Detection systems:

  • Polymer-HRP systems generally provide cleaner backgrounds than ABC

  • DAB as chromogen for brightfield microscopy

  • Consider tyramide signal amplification for low-abundance detection

• Controls and validation:

  • Positive control: Tonsil tissue shows good RAD54L expression

  • Negative controls: Primary antibody omission and isotype controls

  • Peptide competition controls to confirm specificity

  • Compare with known expression patterns from literature and databases

• Scoring and quantification:

  • Establish clear scoring criteria (intensity, percentage positive cells)

  • Consider automated image analysis for consistent quantification

  • Nuclear staining is expected for RAD54L

When developing protocols for tissue microarrays (TMAs), include control tissues on each array and ensure consistent processing conditions across all samples to minimize batch effects.

How can RAD54L antibodies be used in single-cell analysis of DNA repair dynamics?

Single-cell analysis of DNA repair dynamics using RAD54L antibodies represents an emerging frontier in DNA repair research:

• Single-cell immunofluorescence approaches:

  • Quantitative image analysis of RAD54L foci in individual cells

  • Correlation with cell cycle markers (e.g., PCNA patterns, cyclin expression)

  • High-content screening platforms for population-level analysis of single-cell data

  • Time-lapse imaging of fluorescently tagged RAD54L to track dynamics

• Flow cytometry applications:

  • Multi-parameter analysis combining RAD54L with:

    • Cell cycle markers (PI, DAPI)

    • DNA damage markers (γH2AX)

    • Other repair factors (RAD51, BRCA1)

  • Cell sorting for subsequent molecular analysis

  • EdU pulse-chase to correlate with replication status

• Single-cell genomics integration:

  • Combining immunofluorescence with laser capture microdissection

  • INDEX-FACS to link protein expression with single-cell sequencing

  • Analysis of RAD54L expression/localization in correlation with genomic instability markers

• Technical considerations:

  • Signal amplification is often necessary for low abundance proteins

  • Careful fixation to preserve nuclear architecture

  • Balanced permeabilization to maintain cellular integrity while allowing antibody access

  • Multiplexed antibody panels require careful optimization of staining sequences

• Emerging applications:

  • Spatial transcriptomics integration with protein localization

  • Live-cell imaging with antibody fragments (Fabs) or nanobodies

  • Super-resolution microscopy (STORM, PALM) for detailed foci architecture

This approach is particularly valuable for studying heterogeneity in DNA repair capacity within tumors and understanding cell-to-cell variability in response to DNA-damaging therapeutics.

What are the considerations for using RAD54L antibodies in studying alternative lengthening of telomeres (ALT)?

RAD54L has recently been implicated in alternative lengthening of telomeres (ALT), and antibodies against RAD54L can provide valuable insights into this process:

• ALT model systems:

  • U2OS and SAOS-2 are well-established ALT-positive cell lines

  • ALT-positive patient-derived xenografts

  • Isogenic cell line pairs (ALT+ vs. telomerase+)

• Co-localization studies:

  • RAD54L enrichment at ALT telomeres can be detected by co-staining with:

    • TRF1/TRF2 (telomere markers)

    • PML (ALT-associated PML bodies)

    • RAD51 (HR marker at telomeres)

  • Detection of telomeric synthesis through BrdU incorporation

• Functional assays:

  • C-circle assays in correlation with RAD54L levels

  • Telomere ChIP using RAD54L antibodies

  • Combining with telomere FISH to detect RAD54L at telomeres

  • CRISPR-mediated RAD54L knockout/knockdown effects on ALT markers

• Technical optimizations:

  • Pre-extraction protocols to remove soluble nuclear proteins

  • Combining IF with telomere FISH requires careful protocol optimization

  • Methanol fixation sometimes yields better results for nuclear proteins

• Key research findings:

  • RAD54L promotes telomeric DNA synthesis through its ATPase-dependent branch migration activity

  • It plays roles in telomere recombination that are distinct from its functions at DSBs

  • RAD54L is enriched at ALT telomeres specifically

Research has demonstrated that RAD54L's ATPase activity is critical for its function in ALT, suggesting that its branch migration activity may be particularly important in this context .

How can phospho-specific RAD54L antibodies be used to study its regulation?

While phospho-specific RAD54L antibodies are not widely available commercially, this represents an important emerging area for understanding RAD54L regulation:

• Key phosphorylation sites of interest:

  • Serine 49 (S49): Critical for oligomerization and branch migration activity

  • ATPase domain phosphorylation sites: May regulate motor function

  • Cell cycle-dependent phosphorylation sites: Potentially regulate activity during S/G2

• Development strategies:

  • Custom antibody generation against synthetic phosphopeptides

  • Validation in cells treated with phosphatase inhibitors

  • Comparison of signals with/without lambda phosphatase treatment

  • Validation using phospho-mimetic (S→E) and phospho-dead (S→A) mutants

• Research applications:

  • Cell cycle-dependent regulation of RAD54L activity

  • DNA damage-induced phosphorylation changes

  • Kinase inhibitor screens to identify regulatory pathways

  • Correlation of phosphorylation status with functional outcomes

• Analytical approaches:

  • Western blotting with phospho-specific antibodies

  • Immunoprecipitation followed by phospho-antibody detection

  • Mass spectrometry validation of phosphorylation sites

  • Combining with genetic approaches (kinase knockdowns, phospho-mutants)

• Technical considerations:

  • Include phosphatase inhibitors during sample preparation

  • Consider phospho-enrichment methods prior to detection

  • Use appropriate controls (phospho-mimetic mutants, phosphatase treatment)

Understanding RAD54L phosphorylation is particularly relevant given that the S49E mutation (mimicking phosphorylation) causes defects in oligomerization and impacts fork reversal activity , suggesting that phosphorylation at this site may be a regulatory mechanism for RAD54L function in vivo.