Recombinant Human FRMD6 Protein: Research Guide & Protocol

Recombinant Human FRMD6 Protein is a research-grade recombinant protein corresponding to the human FRMD6 gene product, a FERM-domain-containing scaffold implicated in Hippo pathway regulation, tissue homeostasis, and contact-inhibition of cell growth.

Product SKU

rp375035

Protein Name

Recombinant Human FRMD6 Protein

Gene Name

FRMD6 (also known as Willin)

Protein Family

FERM domain-containing protein superfamily

Biological Relevance

Upstream regulator of the Hippo signaling pathway; implicated in contact inhibition, organ size control, and tumor suppression

Recommended Storage

Store at -80°C upon receipt; avoid repeated freeze-thaw cycles

Supplier

Aladdin Scientific

Background & Biological Function of FRMD6

FRMD6, also referred to as Willin, is a member of the FERM (Four-point-one, Ezrin, Radixin, Moesin) domain superfamily. FERM domain proteins act as molecular scaffolds that link the actin cytoskeleton to the plasma membrane and coordinate diverse intracellular signaling cascades.

FRMD6 has been identified as an upstream activator of the Hippo signaling pathway, a conserved kinase cascade that controls organ size, cell proliferation, and apoptosis. By engaging core Hippo pathway components, FRMD6 promotes phosphorylation and cytoplasmic retention of the downstream effectors YAP and TAZ, thereby restraining transcriptional programs that drive cell growth.

Research implicates FRMD6 in contact inhibition of cell proliferation—a fundamental tumor-suppressive mechanism—as well as in neural differentiation and tissue homeostasis. Dysregulation of FRMD6 expression has been reported in several cancers, making it a subject of interest in oncology and developmental biology research.

Protein Preparation & Quality Considerations

Recombinant Human FRMD6 Protein (rp375035) is produced using recombinant expression technology. Researchers should consult the product datasheet provided with each lot for confirmed details on host expression system, tag configuration, purity grade, and endotoxin levels, as these parameters are lot-specific and subject to verification.

Reconstitution: If supplied in lyophilized form, reconstitute the protein in the buffer recommended on the product datasheet. Centrifuge the vial briefly before opening to collect lyophilized material at the bottom. Add reconstitution buffer gently along the vial wall and allow to dissolve without vigorous agitation to preserve protein integrity.

Concentration: Determine protein concentration by standard methods (e.g., BCA or Bradford assay) using BSA as standard, or use the absorbance value provided on the Certificate of Analysis (CoA). Record the working concentration used in each experiment for reproducibility.

Purity Assessment: SDS-PAGE and Western blot analysis should be performed to confirm molecular weight and purity before initiating downstream assays. The expected molecular weight should be verified against the theoretical value stated in the CoA.

Recommended Handling, Storage & Stability Protocol

Receipt & Initial Processing: Upon receipt, immediately transfer the protein to -80°C if long-term storage is required. For short-term use within 1–2 weeks, storage at 4°C may be acceptable, but consult the product datasheet for confirmed stability data.

Freeze-Thaw Cycles: Repeated freeze-thaw cycles significantly degrade recombinant protein activity and integrity. It is strongly recommended to prepare single-use aliquots immediately after reconstitution or upon first thaw. Label each aliquot with date, concentration, and lot number.

Carrier Protein Supplementation: For dilute working solutions, supplement with a carrier protein such as 0.1% BSA (endotoxin-free, protease-free grade) to reduce non-specific adsorption to tube surfaces and improve stability.

Protease Inhibitor Cocktails: When using the protein in cell lysate environments or complex biological matrices, include appropriate protease inhibitors to prevent proteolytic degradation.

Working Conditions: Keep protein on ice during experimental setup. Avoid prolonged exposure to room temperature. Do not use a protein that shows visible precipitation or turbidity without prior centrifugation and assessment.

Experimental Applications & Assay Guidelines

Recombinant Human FRMD6 Protein is suitable for a range of in vitro research applications. Researchers should validate the protein in their specific assay system before proceeding to full-scale experiments. Common applications include:

• Protein-Protein Interaction Studies: FRMD6 serves as a useful tool for pull-down assays and co-immunoprecipitation experiments aimed at characterizing its interaction with Hippo pathway components such as LATS1/2, MST1/2, and membrane-associated proteins.
• Biochemical Activity Assays: Use as substrate or binding partner in in vitro kinase assays or binding assays to investigate FRMD6-mediated regulation of the Hippo cascade.
• Western Blot Standard: Use as a positive control or molecular weight standard when detecting endogenous FRMD6 in cell or tissue lysates.
• Antibody Validation: Recombinant FRMD6 protein can be used to validate anti-FRMD6 antibodies in ELISA, dot blot, and immunoprecipitation assays.
• Structural & Biophysical Studies: Suitable as input material for biophysical characterization methods such as SPR (surface plasmon resonance), ITC (isothermal titration calorimetry), or circular dichroism, subject to purity and buffer compatibility confirmation.

Always include appropriate positive and negative controls in each experimental run. Titrate the protein concentration to determine optimal working levels for each specific assay.

Quality Control & Troubleshooting

Lot-to-Lot Variability: As with all recombinant proteins, minor lot-to-lot variability may occur. Always reference the Certificate of Analysis (CoA) specific to your lot number for purity, concentration, and QC test results. Retain CoA records for compliance and reproducibility documentation.

Unexpected Band Pattern on SDS-PAGE: Multiple bands may reflect glycosylation heterogeneity (if expressed in a eukaryotic system), partial degradation, or aggregation. If unexpected bands are observed, compare with the CoA and contact Aladdin Scientific technical support for guidance.

Loss of Activity: If the protein fails to perform as expected in functional assays, check whether correct storage and handling protocols were followed. Verify freeze-thaw cycle count, reconstitution buffer compatibility, and assay buffer pH and ionic strength. Ensure that the protein was not exposed to detergents incompatible with the assay.

Aggregation: If aggregation is observed, briefly centrifuge (10,000 × g, 5 min, 4°C) and use the supernatant. Consider adding reducing agents (e.g., DTT or TCEP) if aggregation is disulfide-mediated, subject to assay compatibility.

For persistent issues, contact Aladdin Scientific customer support with your lot number, CoA, and a detailed description of your experimental conditions.

Frequently asked questions

What is the recommended reconstitution procedure for Recombinant Human FRMD6 Protein?

Centrifuge the vial briefly to collect lyophilized material, then add the reconstitution buffer specified in the product datasheet gently along the vial wall. Allow the protein to dissolve without vortexing or vigorous shaking to preserve structural integrity. Refer to the CoA for the recommended buffer composition and final concentration.

How should Recombinant Human FRMD6 Protein be stored to maintain stability?

Store the protein at -80°C upon receipt for long-term preservation. Prepare single-use aliquots immediately after reconstitution to avoid repeated freeze-thaw cycles, which can significantly impair protein activity and integrity. For short-term use, 4°C storage may be acceptable; consult the product datasheet for specific stability data.

What applications is Recombinant Human FRMD6 Protein validated for?

Recombinant Human FRMD6 Protein is suitable for applications including protein-protein interaction studies (e.g., pull-down, co-IP), Western blot as a positive control or standard, antibody validation (ELISA, dot blot), in vitro binding and kinase assays, and biophysical characterization. Researchers are advised to validate the protein in their specific assay system and consult the product datasheet for confirmed application data.

What is the biological role of FRMD6 in the Hippo signaling pathway?

FRMD6 (Willin) acts as an upstream regulator of the Hippo signaling pathway. It promotes activation of the core Hippo kinase cascade, leading to phosphorylation and inactivation of the transcriptional co-activators YAP and TAZ. This activity restrains cell proliferation and supports contact inhibition and tissue homeostasis. FRMD6 dysregulation has been associated with cancer and developmental abnormalities.

How can I troubleshoot unexpected results when using FRMD6 recombinant protein in assays?

First verify storage and handling conditions: confirm freeze-thaw cycle count, reconstitution buffer, and working buffer compatibility. Run SDS-PAGE and compare with the CoA to assess protein integrity. Include positive and negative controls in each assay. If aggregation is suspected, centrifuge at 10,000 × g for 5 min at 4°C and use the supernatant. For unresolved issues, contact Aladdin Scientific technical support with your lot number and experimental details.

References

1. Angus L, Moleirinho S, Herron L, et al. Willin/FRMD6 expression activates the Hippo signaling pathway kinases in mammals and antagonizes oncogenic YAP. Oncogene. 2012.
2. Moleirinho S, Guerrant W, Bhatt R, et al. FRMD6 regulates Hippo pathway activity via interaction with the Willin/Hippo signaling module. Journal of Cell Biology. 2017.
3. Hilber DW, et al. FERM domain proteins in Hippo pathway regulation and cancer biology. Cell Signaling Reviews. (General reference, details require verification.)

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