RFXANK Antibody, Biotin conjugated

What is RFXANK and why is it significant in immunological research?

RFXANK (Regulatory Factor X-Associated Ankyrin Containing Protein) is a critical component of the heterotrimeric RFX complex, which also includes RFX5 and RFXAP. This complex plays an essential role in regulating Major Histocompatibility Complex (MHC) class II gene expression by binding to the X1 box of MHC-II promoters .

RFXANK is particularly significant in immunological research because:

• It activates transcription from class II MHC promoters, requiring the activity of MHC class II transactivator (CIITA)

• Mutations in the RFXANK gene are associated with Bare Lymphocyte Syndrome (BLS), a rare autosomal recessive form of combined immunodeficiency

• Approximately two-thirds of all MHC class II deficiency cases result from autosomal-recessive mutations in the RFXANK gene

• The protein contains ankyrin repeats (particularly in domains 1-4) that are crucial for protein-protein interactions

Recent studies have also revealed its interaction with caspase-2, suggesting potential non-apoptotic roles in MHC class II gene regulation .

What applications are biotin-conjugated RFXANK antibodies suitable for?

Biotin-conjugated RFXANK antibodies are versatile tools suitable for multiple research applications:

When selecting a biotin-conjugated RFXANK antibody, consider the specific clone and validate it for your particular application. For example, the OTI3B4 clone (catalog numbers like ABIN1500684) has been validated for WB and IF applications with human samples .

What is the typical molecular weight observed for RFXANK in Western blot analysis?

Researchers should expect to observe RFXANK at different molecular weights depending on the isoform and post-translational modifications:

• Calculated molecular weight: 28.1-28.2 kDa

• Observed molecular weight: ~34 kDa in most experimental conditions

• The full-length protein (NP_604389) produced in HEK293T cells is commonly used as a positive control

It's worth noting that discrepancies between calculated and observed molecular weights are common with many proteins due to post-translational modifications or the presence of different isoforms. When running Western blots for RFXANK, include appropriate positive controls such as RFXANK-expressing HEK293T cell lysates .

What are the recommended protocols for using biotin-conjugated RFXANK antibodies in flow cytometry?

For optimal results when using biotin-conjugated RFXANK antibodies in flow cytometry:

Protocol Overview:

• Harvest and wash cells in cold PBS containing 2% FBS

• Fix cells (if required) with 4% paraformaldehyde for 15 minutes at room temperature

• For intracellular staining, permeabilize with 0.1% Triton X-100 in PBS for 5 minutes

• Block with 5% normal serum in PBS for 30 minutes

• Incubate with biotin-conjugated RFXANK antibody at 1:100 dilution for 1 hour at room temperature

• Wash 3 times with PBS containing 2% FBS

• Incubate with streptavidin-conjugated fluorophore for 30 minutes at room temperature

• Wash 3 times and analyze by flow cytometry

Critical Considerations:

• Include appropriate isotype controls (such as mouse IgG2a or IgG2b depending on the antibody clone)

• For clone OTI3E7, a dilution of 1:100 is recommended for flow cytometry applications

• Always titrate the antibody for optimal signal-to-noise ratio

• For intracellular RFXANK detection, permeabilization is required as the protein is primarily located in the cytoplasm

How should storage and handling of biotin-conjugated RFXANK antibodies be optimized?

Proper storage and handling are critical for maintaining antibody activity:

Long-term storage recommendations:

• Store at -20°C or -80°C in small aliquots (no less than 20 μl) to avoid freeze-thaw cycles

• Some antibodies can be stored at 4°C for up to two weeks for immediate use

• Avoid repeated freeze-thaw cycles as they degrade antibody quality

Handling recommendations:

• For lyophilized antibodies (e.g., ABIN1500684), reconstitute by adding 0.2 ml of distilled water to yield a concentration of 500 μg/ml

• After reconstitution, antibodies can typically be stored at 4°C for one month

• Some preparations contain preservatives like 0.05% sodium azide, 0.02% ProClin, or 1% BSA with 50% glycerol

Specific example:
Fisher Scientific's biotin-conjugated RFXANK antibody (OTI3B4) is formulated in PBS with 0.05% sodium azide and should be stored at 4°C in the dark for short-term use or aliquoted and stored at -20°C for long-term storage .

What controls should be included when using biotin-conjugated RFXANK antibodies in immunofluorescence studies?

To ensure reliable and interpretable results in immunofluorescence studies, include these essential controls:

Positive controls:

• Cell lines known to express RFXANK (HEK293T cells with recombinant RFXANK expression)

• B lymphocytes (which naturally express RFXANK as part of MHC class II regulation)

Negative controls:

• RFXANK-deficient cell lines (such as BLS-1, a RFXANK-deficient cell line with null mutation)

• Secondary antibody alone (omitting primary antibody)

• Isotype control (matching the primary antibody's isotype, such as mouse IgG2a or IgG2b)

Methodology controls:

• Blocking peptide competition assay to confirm specificity

• Dual staining with another RFXANK antibody targeting a different epitope

• Nuclear counterstain (such as DAPI) to visualize cellular context

For optimal specificity validation, compare staining patterns between wild-type cells and RFXANK-silenced cells (using RNAi or CRISPR)—the latter should show significantly reduced signal intensity.

How can biotin-conjugated RFXANK antibodies be used to study RFXANK-protein interactions?

Biotin-conjugated RFXANK antibodies are valuable tools for investigating protein-protein interactions:

Co-immunoprecipitation (Co-IP) protocol:

• Prepare cell lysates in a buffer containing 20 mM HEPES, 100 mM KCl, 0.5 mM DTT, 0.1% BSA, 0.1% NP-40, and protease inhibitors

• Pre-clear lysates with protein A/G beads

• Incubate lysates with biotin-conjugated RFXANK antibody (5-10 μg) for 2-4 hours at 4°C

• Add streptavidin-conjugated magnetic beads and incubate for 1 hour

• Wash beads 3-5 times with wash buffer

• Elute bound proteins and analyze by SDS-PAGE followed by Western blotting

Known interactions to investigate:

• RFX5 and RFXAP (which form the heterotrimeric RFX complex with RFXANK)

• CIITA (class II transactivator)

• Caspase-2 (recently identified interaction partner)

• ANKRA2 (a related protein that can functionally replace the ARD of RFXANK)

Research by Kotsias et al. demonstrated that the ankyrin repeat domain of RFXANK (particularly repeats 1-3) is sufficient for interaction with proteins like caspase-2, with co-localization occurring primarily in the cytoplasm .

How do mutations in the RFXANK gene affect antibody recognition and experimental outcomes?

Mutations in the RFXANK gene can significantly impact antibody recognition and experimental results:

Common mutations and their effects:

• The 752delG26 mutation (I5E6-25_I5E6+1) is a 26-bp deletion found predominantly in North African populations

• Mutations in the fourth ankyrin repeat can abolish assembly of the enhanceosome on MHC-II promoters in vivo but not in vitro

• Mutations within ankyrin repeats 1 and 3 interfere with formation of the RFX complex

Experimental considerations:

• Antibodies targeting epitopes within mutated regions may show reduced or absent binding

• For populations with known high prevalence of specific mutations (e.g., North African populations with 752delG26), select antibodies targeting conserved regions

• When studying patient samples with suspected RFXANK mutations, use multiple antibodies targeting different epitopes

Detection strategy:
When working with samples that might contain RFXANK mutations, use antibodies targeting the N-terminal region (amino acids 1-90), which is often preserved in many mutations, alongside antibodies targeting the ankyrin repeat domain .

What are the critical differences in using biotin-conjugated versus unconjugated RFXANK antibodies in multi-color flow cytometry?

Understanding the advantages and limitations of biotin conjugation is essential for experimental design:

Advantages of biotin-conjugated antibodies:

• Signal amplification through multiple streptavidin-fluorophore binding to each biotin molecule

• Flexibility to use different streptavidin conjugates (PE, APC, FITC) with the same primary antibody

• Useful for detecting low-abundance proteins like RFXANK

• Can be combined with directly conjugated antibodies in multi-color panels

Limitations and considerations:

• Additional incubation step required for streptavidin-fluorophore binding

• Potential background from endogenous biotin in certain tissues/cells

• Possible blocking of nearby epitopes due to streptavidin size

• Biotin-streptavidin interactions may be affected by certain fixation methods

Panel design recommendations:
When designing multi-color panels including biotin-conjugated RFXANK antibodies:

• Use biotin blocking reagents if samples contain high endogenous biotin

• Reserve brightest fluorochromes (PE, APC) for low-abundance targets like RFXANK

• Conduct compensation controls using the same streptavidin-fluorophore conjugate

• Consider the spectral overlap between fluorochromes when selecting streptavidin conjugates

For intracellular RFXANK detection alongside surface markers, perform surface staining first, followed by fixation, permeabilization, and then intracellular staining with the biotin-conjugated RFXANK antibody.

How can researchers distinguish between specific and non-specific binding when using biotin-conjugated RFXANK antibodies?

To ensure reliable data interpretation, implement these strategies to distinguish specific from non-specific binding:

Validation approaches:

• Blocking experiments: Pre-incubate the antibody with recombinant RFXANK protein before staining. Specific binding should be significantly reduced.

• RFXANK-deficient controls: Compare staining between wild-type cells and RFXANK-knockout or knockdown cells. Specific signals should be absent or significantly reduced in the latter.

• Antibody titration: Perform a dilution series to identify the optimal concentration where specific signal is maintained while background is minimized.

• Isotype controls: Use matched isotype controls (e.g., mouse IgG2a or IgG2b) at the same concentration as the primary antibody .

Expected staining patterns:

• Cytoplasmic localization is expected for RFXANK

• In co-immunoprecipitation experiments with endogenous proteins, RFXANK-caspase-2 binding occurs predominantly in the cytoplasm

• Molecular weight verification in Western blots (approximately 34 kDa)

If non-specific binding is observed, try increasing blocking agent concentration (5-10% serum or BSA), reducing primary antibody concentration, or including additional washing steps in your protocol.

What are common technical issues when using biotin-conjugated RFXANK antibodies and how can they be addressed?

For optimal immunoprecipitation of RFXANK-interacting proteins, follow the protocol established by Kotsias et al., which uses a binding buffer containing 20 mM HEPES, 100 mM KCl, 0.5 mM DTT, 0.1% BSA, 0.1% NP-40, and protease inhibitors .

How can researchers quantify RFXANK expression levels accurately using biotin-conjugated antibodies?

Accurate quantification of RFXANK requires appropriate methods and controls:

Western blot quantification:

• Use recombinant RFXANK standards at known concentrations to generate a standard curve

• Include housekeeping protein controls (β-actin, GAPDH) for normalization

• Ensure linear range detection by testing multiple sample dilutions

• Use digital imaging software for densitometry analysis

• Present data as relative expression normalized to control samples

Flow cytometry quantification:

• Use calibration beads with known binding capacity to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

• Include a standard cell line with stable RFXANK expression as an inter-assay control

• Report data as median fluorescence intensity (MFI) or MESF

• For comparing patient samples, calculate the ratio of patient to control MFI

ELISA-based quantification:

• Develop a sandwich ELISA using a capture antibody against one RFXANK epitope and biotin-conjugated detection antibody against a different epitope

• Generate a standard curve using recombinant RFXANK

• Measure optical density and interpolate unknown sample concentrations

For precise quantification in clinical samples, consider that RFXANK expression may vary in different cell types and can be altered in disease states such as Bare Lymphocyte Syndrome . In molecular investigations of MHC class II deficiency, correlate RFXANK protein levels with MHC class II expression and patient phenotypes.

How can biotin-conjugated RFXANK antibodies be used to study MHC class II deficiency syndromes?

Biotin-conjugated RFXANK antibodies are valuable tools for investigating MHC class II deficiency (Bare Lymphocyte Syndrome):

Clinical research applications:

• Diagnostic screening: Detect RFXANK protein expression in peripheral blood mononuclear cells from suspected cases

• Mutation characterization: Assess how specific mutations affect antibody binding to different RFXANK epitopes

• Genotype-phenotype correlation: Compare RFXANK expression levels with the severity of MHC class II deficiency

Experimental approach for patient samples:

• Isolate peripheral blood mononuclear cells (PBMCs) from patients and controls

• Perform flow cytometry using biotin-conjugated RFXANK antibodies alongside MHC class II (HLA-DR, HLA-DP, HLA-DQ) markers

• Analyze correlation between RFXANK expression and MHC class II surface expression

Research findings:
Studies have shown that the 752delG26 mutation in RFXANK is particularly common in North African populations, accounting for approximately two-thirds of all MHC class II deficiency cases . This founder mutation, dated to approximately 2250 years ago (95% CI: 1750-3025 years), has significant implications for genetic counseling and molecular diagnosis in affected families .

When investigating patient samples, researchers should be aware that some mutations may preserve protein expression while disrupting function, necessitating functional assays alongside expression analysis.

What methodological approaches can be used to study RFXANK-caspase-2 interactions in immune regulation?

The recently discovered interaction between RFXANK and caspase-2 opens new avenues for investigating non-apoptotic functions in immune regulation :

Co-localization studies:

• Perform dual immunofluorescence with biotin-conjugated RFXANK antibody and fluorescently labeled caspase-2 antibody

• Use confocal microscopy to examine subcellular localization

• Quantify co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

Functional investigation methods:

• Domain mapping: Utilize truncated RFXANK constructs (especially ankyrin repeats 1-3) to identify critical interaction domains

• Mutagenesis: Introduce point mutations in RFXANK ankyrin repeats to disrupt specific interactions

• Protein-protein interaction dynamics: Use proximity ligation assay (PLA) to visualize and quantify endogenous RFXANK-caspase-2 interactions in situ

Experimental findings:
Research has demonstrated that:

• The interaction between caspase-2 and RFXANK occurs primarily in the cytoplasm

• The ankyrin repeat domain (particularly repeats 1-3) is sufficient for protein interaction

• RFXANK overexpression enhances caspase-2 processing in cells treated with chemotherapeutic agents

• Caspase-2 may have a non-apoptotic role in MHC class II gene regulation, as evidenced by increased total MHC class II protein levels in caspase-2-silenced cells