Recombinant Human CHFR Protein: Key FAQs & Usage Guide

Recombinant Human CHFR Protein is a research-grade recombinant form of the human Checkpoint with Forkhead-associated and RING domains (CHFR) protein, produced for use in biochemical assays, ubiquitination studies, and cell cycle checkpoint research.

Product Name

Recombinant Human CHFR Protein

SKU

rp365382

Protein Function

E3 ubiquitin ligase involved in the antephase checkpoint, mediating mitotic stress response

Key Domains

Forkhead-associated (FHA) domain and RING finger domain

Research Relevance

CHFR is frequently silenced by promoter methylation in various human cancers, making it a candidate tumor suppressor

Typical Applications

In vitro ubiquitination assays, protein interaction studies, Western blot, functional checkpoint assays

What Is CHFR and Why Is It Important in Research?

CHFR (Checkpoint with Forkhead-associated and RING domains) is a human E3 ubiquitin ligase that plays a critical role in the antephase checkpoint — a mitotic stress-response pathway that delays entry into mitosis when cells are exposed to microtubule poisons or other mitotic stressors. The protein contains two key functional domains: an N-terminal Forkhead-associated (FHA) domain, which is involved in phosphopeptide binding and protein–protein interactions, and a C-terminal RING finger domain, which confers E3 ubiquitin ligase activity.

CHFR has attracted significant research interest because its expression is frequently lost in a wide range of human cancers — including colorectal, gastric, esophageal, and lung cancers — most often through epigenetic silencing via CpG island promoter hypermethylation. This pattern of inactivation suggests that CHFR functions as a tumor suppressor. Consequently, Recombinant Human CHFR Protein is a valuable tool for researchers studying ubiquitin-mediated protein degradation, mitotic checkpoint mechanisms, and cancer biology.

What Are the Recommended Storage and Handling Conditions?

Proper storage and handling of Recombinant Human CHFR Protein is essential to maintain its activity and integrity. As with most recombinant proteins, general best practices include the following recommendations — however, researchers should always refer to the specific Certificate of Analysis (CoA) and product datasheet supplied with SKU rp365382 for confirmed, lot-specific conditions:

• Short-term storage: Store at 4°C for up to one week if the protein will be used promptly.
• Long-term storage: Aliquot and store at −20°C or −80°C to avoid repeated freeze–thaw cycles, which can degrade protein activity.
• Reconstitution: If supplied lyophilized, reconstitute in the buffer specified in the datasheet (typically sterile PBS or Tris-based buffer) to the recommended concentration. Allow to equilibrate at room temperature briefly before use.
• Additives: A carrier protein such as 0.1% BSA may be recommended for dilute solutions to prevent adsorption to tube surfaces.

Always handle on ice during experimental setup. Avoid vortexing; mix gently by pipetting or brief centrifugation. Contact Aladdin Scientific technical support if specific storage guidance is required for your application.

What Applications Is Recombinant Human CHFR Protein Suitable For?

Recombinant Human CHFR Protein (rp365382) is suitable for a range of in vitro and biochemical research applications, including but not limited to:

• In vitro ubiquitination assays: CHFR's E3 ligase activity can be assessed in reconstituted ubiquitination systems containing E1 activating enzyme, E2 conjugating enzyme, ubiquitin, and ATP. This enables mechanistic studies of substrate ubiquitination and subsequent proteasomal degradation.
• Protein–protein interaction studies: The FHA and RING domains of CHFR interact with multiple partners. Pull-down assays, co-immunoprecipitation, and surface plasmon resonance experiments can be performed using the recombinant protein to map interaction interfaces.
• Western blot (positive control): The recombinant protein serves as a molecular weight marker and positive control for antibody validation in immunoblotting experiments.
• Enzyme activity profiling: Kinetic studies and inhibitor screening targeting CHFR's E3 ligase function can be performed with defined recombinant protein preparations.
• Structural and biophysical studies: The purified protein may support crystallography, cryo-EM, or biophysical characterization experiments depending on the supplied formulation and purity.

For application-specific optimization, consult the product datasheet or contact Aladdin Scientific technical support.

Quality, Purity, and Formulation Considerations

Research-grade recombinant proteins must meet defined quality standards to ensure reproducible experimental results. For Recombinant Human CHFR Protein (rp365382), researchers should review the product-specific Certificate of Analysis (CoA) for the following parameters:

• Purity: Typically assessed by SDS-PAGE and/or HPLC. Research-grade recombinant proteins generally target ≥85–95% purity, though the confirmed value for each lot is stated in the CoA.
• Biological activity: Functional activity may be confirmed by an in vitro ubiquitination assay or equivalent functional test, as detailed in the product datasheet.
• Endotoxin level: Low endotoxin content is critical for cell-based assays. Endotoxin levels are typically measured by the Limulus Amebocyte Lysate (LAL) method and reported in EU/µg.
• Formulation: The protein is typically supplied in a stabilizing buffer, which may contain glycerol or other cryoprotectants. The exact formulation is lot-specific and documented in the CoA.
• Tag information: The recombinant protein may carry an affinity tag (e.g., His-tag or GST-tag) used during purification. Tag presence and position are specified in the product datasheet and should be considered when designing assays.

If your application requires specific purity thresholds or endotoxin limits, contact Aladdin Scientific before ordering to confirm lot suitability.

Frequently asked questions

What is the biological function of CHFR protein?

CHFR is an E3 ubiquitin ligase that acts at the antephase checkpoint to delay mitotic entry in response to microtubule stress. It contains an FHA domain for protein interactions and a RING domain that catalyzes ubiquitin transfer to target substrates, marking them for proteasomal degradation. Loss of CHFR expression is associated with checkpoint deficiency and is commonly observed in several human cancers.

How should I determine the correct working concentration of Recombinant Human CHFR Protein for my assay?

The optimal working concentration depends on your specific assay format and experimental conditions. For in vitro ubiquitination assays, a titration experiment is recommended to identify the minimum protein amount that yields a detectable and reproducible signal. Please consult the product datasheet for any recommended starting concentrations provided by Aladdin Scientific, and contact technical support if further guidance is needed.

Is Recombinant Human CHFR Protein suitable for use in cell-based assays?

Recombinant Human CHFR Protein is primarily designed for in vitro biochemical applications. Use in cell-based assays may be possible but requires validation of endotoxin levels and formulation compatibility with your cell system. We recommend confirming endotoxin content from the CoA and, if necessary, requesting a low-endotoxin preparation. Contact Aladdin Scientific technical support to discuss your specific application requirements.

Why is CHFR considered a tumor suppressor, and how is this relevant to cancer research?

CHFR is considered a tumor suppressor because its expression is frequently silenced by CpG island promoter hypermethylation across multiple cancer types, including colorectal, gastric, esophageal, and lung cancers. Loss of CHFR impairs the antephase checkpoint, allowing cells with mitotic defects to progress through division, which can promote genomic instability. Recombinant Human CHFR Protein enables researchers to conduct mechanistic studies of this tumor suppressor pathway and evaluate potential therapeutic strategies targeting the CHFR-ubiquitin axis.

What should I do if the recombinant protein does not perform as expected in my assay?

First, verify storage and handling conditions, reconstitution buffer, and protein concentration against the product datasheet. Ensure that all assay components (e.g., E1, E2 enzymes, ATP, and ubiquitin for ubiquitination assays) are functional and within their working concentration ranges. If the issue persists, contact Aladdin Scientific customer or technical support with your lot number, CoA details, and a description of your experimental setup for troubleshooting assistance.

References

1. Scolnick DM, Halazonetis TD. Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature. 2000;406(6794):430-435. doi:10.1038/35019108
2. Mizuno K, et al. Epigenetic inactivation of CHFR in human colorectal cancer. Oncogene. 2002;21(23):3810-3813.
3. Mariappan MM, et al. CHFR: Gatekeeper of the antephase checkpoint. Cell Cycle. 2007;6(10):1166-1171.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.