RFX5 Antibody

Basic Research Applications

• What is the function of RFX5 protein and why is it significant in research?

  RFX5 functions as a key transcription regulator that activates transcription from class II MHC promoters and recognizes X-boxes. It mediates cooperative binding between RFX and NF-Y, with RFX binding to the X1 box of MHC-II promoters . RFX5 plays a crucial role in the immune system through regulation of MHCII gene expression . Notably, deficiencies or malfunctions in RFX5 have strong connections with Bare Lymphocyte Syndrome (BLS) type II, characterized by reduced expression of MHC class II molecules . Recent research has also identified RFX5 as a significant factor in hepatocellular carcinoma (HCC) progression through transcriptionally activating KDM4A expression .

• What are the most common applications for RFX5 antibodies in laboratory research?

  RFX5 antibodies are utilized across multiple laboratory techniques:

  Application	Typical Dilution Range	Notes
  Western Blotting (WB)	1:500-1:3000	Most commonly validated application
  ELISA	1:5,000-1:25,000	High dilution range suggests sensitivity
  Immunofluorescence (IF)	Varies by antibody	Effective for localization studies
  ChIP	0.5-1.0 μl per assay	Used for studying DNA-protein interactions
  Immunoprecipitation (IP)	2.0 μg per 500 μg extract	Effective for protein complex studies
  Gel supershift assays	0.5-1.0 μl per assay	Validated for RFX5 complex detection

• What are the critical considerations for storing and handling RFX5 antibodies?

  Proper storage and handling of RFX5 antibodies is essential for maintaining reactivity:

  • Store unopened vials at -20°C

  • For long-term storage, aliquot contents and freeze at -20°C or below to avoid repeated freeze-thaw cycles

  • Thawed antibodies can remain stable for several weeks at 4°C as undiluted liquid

  • Centrifuge product if not completely clear after standing at room temperature

  • Dilute only immediately before use

  • Expiration is typically one year from opening date when properly stored

  • Some formulations contain sodium azide (0.01-0.02%) as a preservative

Technical Troubleshooting

• What are the common technical challenges when using RFX5 antibodies and how can they be addressed?

  Common challenges and solutions:

  • Multiple bands on Western blot: Documented minor bands at 65 kDa and 80 kDa may appear with some antibodies . Increase blocking time/concentration or try alternative blocking agents (5% BLOTTO overnight at 4°C has shown good results) .

  • Weak signal: Optimize primary antibody concentration (1:500-1:1000 rather than 1:3000), increase incubation time (overnight at 4°C), or use more sensitive detection methods.

  • High background: Increase washing steps, optimize blocking conditions, and ensure secondary antibody is highly specific to the primary antibody host species.

  • Sample preparation: For optimal RFX5 detection, use nuclear extracts as RFX5 is predominantly nuclear . Ensure proteins are not degraded during extraction by using appropriate protease inhibitors.

  • Storage-related issues: Avoid repeated freeze-thaw cycles by aliquoting the antibody. Some RFX5 antibodies have shown stability at 4°C for weeks as undiluted liquid .

  • BSA interference: For certain applications, BSA-free formulations may be required. Some manufacturers can provide BSA-free versions upon request .

• What are the optimal protein extraction methods for studying RFX5 expression in different cellular compartments?

  For effective RFX5 protein extraction:

  • Nuclear extraction: Since RFX5 is a nuclear transcription factor, use dedicated nuclear extraction protocols. Successful detection has been reported using nuclear extract lysates (10 μg per lane) .

  • S100 cytosolic fraction: For specific experiments studying RFX5 degradation, dounce homogenization to make the cytosolic fraction S100 has been effective .

  • Proteasome inhibition: When studying RFX5 degradation, consider including proteasome inhibitors in extraction buffers as RFX5 can undergo proteasomal degradation .

  • Buffer composition: Use buffers containing potassium phosphate (0.02 M) and sodium chloride (0.15 M) at pH 7.2 for antibody stability .

  • Protease inhibitors: Always include freshly prepared protease inhibitor cocktail in extraction buffers to prevent degradation.

  • Sample handling: Process samples quickly and keep them cold throughout extraction to minimize protein degradation.

Advanced Research Topics

• How can RFX5 antibodies be utilized in studying the RFX5-KDM4A pathway in cancer progression?

  To investigate the RFX5-KDM4A pathway:

  • ChIP-PCR assay: Use RFX5 antibodies for ChIP followed by PCR with primers designed to detect the RFX5 binding peak site in the KDM4A promoter region. This approach has successfully demonstrated direct binding of RFX5 to the KDM4A promoter in SK-HEP-1, MHCC-97H, and HepG2 cells .

  • Luciferase reporter assay: Combine with KDM4A promoter constructs to measure the impact of RFX5 on KDM4A transcription.

  • Western blot analysis: Use both RFX5 and KDM4A antibodies to monitor their expression levels in response to RFX5 overexpression or knockdown. Research shows RFX5 overexpression elevated KDM4A protein levels, while RFX5 knockdown downregulated KDM4A .

  • qRT-PCR: Monitor mRNA levels of both RFX5 and KDM4A to establish transcriptional regulation.

  • Cell cycle analysis: Use flow cytometry with 7-AAD and BrdU staining to examine how RFX5-KDM4A pathway affects cell cycle progression .

  • Rescue experiments: Perform KDM4A overexpression in RFX5-depleted cells to demonstrate pathway dependencies .

• What techniques can be employed to study RFX5 degradation during cellular processes?

  To investigate RFX5 degradation:

  • Time course experiments: Monitor RFX5 levels at various time points during cellular processes. Research has shown correlation between infection duration and RFX5 degradation .

  • Proteasome inhibition: Use proteasome inhibitors (MG132, lactacystin) to determine if RFX5 degradation is proteasome-dependent.

  • Immunoprecipitation: Use RFX5 antibodies for IP followed by ubiquitin detection to assess ubiquitination status.

  • Cell-free degradation assay: Utilize S100 cytosolic fractions with or without proteasome depletion to study RFX5 degradation mechanisms .

  • Antibody depletion experiments: Use antibodies against proteasome components (e.g., mAb MCP21 specific to 20S proteasome α subunit) to deplete proteasome complexes and assess their role in RFX5 degradation .

  • Two-dimensional PAGE analysis: Compare RFX5 protein modifications under normal versus degradation-inducing conditions .

• How can RFX5 antibodies be used to investigate the relationship between RFX5 and p53 signaling in cancer?

  To study RFX5-p53 interactions:

  • Co-immunoprecipitation: Use RFX5 antibodies to pull down associated proteins, followed by Western blot for p53 to detect potential interactions.

  • Western blot analysis: Monitor levels of p53, p21, and Bax in response to RFX5 manipulation. Research has shown RFX5 overexpression significantly suppressed p53, p21, and Bax expression, while RFX5 silencing upregulated these proteins .

  • ChIP-seq analysis: Use RFX5 antibodies for ChIP-seq to identify genome-wide binding sites and analyze overlap with p53 target genes.

  • Dual reporter assays: Measure p53 transcriptional activity in the presence of varying RFX5 levels.

  • Immunofluorescence: Use antibodies against both RFX5 and p53 to examine their cellular localization and potential co-localization.

  • Cell cycle and apoptosis assays: Combine with flow cytometry to assess how RFX5-mediated regulation of p53 affects cell cycle progression and apoptosis rates.