Key Features
- Detects Mouse RASSF6 with High Specificity: No cross-reactivity observed with other mouse RASSF family members (RASSF1, 2, 3, 4, 5) or Ras superfamily proteins (Ras, Rap). Designed for unambiguous quantitation in complex biological matrices.
- Sensitive pg/mL Range: Quantitative range 1.0–200 pg/mL, minimum detectable concentration < 1.0 pg/mL, standard curve from 0 to 200 pg/mL (6 points).
- Recombinant Protein Standard: Produced in a eukaryotic expression system for native-like conformation and optimal antibody recognition.
- Validated for Multiple Sample Types: Mouse serum, EDTA/citrate/heparin plasma, cell culture supernatant, and tissue homogenates.
- Optimized Tissue & Cell Extraction: Protocols include mandatory use of protease inhibitors, recommended RIPA or mild detergent buffers (Triton X-100, NP-40), and low-temperature processing to maintain RASSF6 integrity.Select the appropriate cell lysis solution to avoid any influence of the lysis solution on the experimental results.
- Built-in 5× Sample Dilution: The assay automatically dilutes samples 5-fold in the well (10 µL sample + 40 µL diluent). Multiply the interpolated result by 5 to obtain the actual concentration.
- One-Step Sandwich ELISA: Simultaneous incubation of sample/standard and HRP-detection antibody; total assay time approximately 2 hours.
- Reliable Precision: Intra- and inter-assay CV < 15%; standard curve linearity R ≥ 0.9900.
- Flexible Formats: 48-well and 96-well plates with removable strips; up to 88 samples per plate (single wells).
- Stable Storage: 6-month shelf life at 2–8°C, protected from light and moisture; cold-chain shipping.
Product Description
RASSF6 (RAS association domain-containing protein 6) is a member of the RASSF family of tumor suppressors and scaffold proteins. It plays critical roles in regulating the Hippo signaling pathway, apoptosis, cell cycle arrest, and inflammation. Loss of RASSF6 expression has been implicated in various cancers and tissue injury models. In mouse research, accurate quantitation of RASSF6 is essential for mechanistic studies in oncology, immunology, and developmental biology.
The Yanda-MOUSE-RASSF6 ELISA Kit employs a one-step double-antibody sandwich ELISA. A pre-coated capture antibody binds mouse RASSF6, and an HRP-conjugated detection antibody is added simultaneously. After washing, TMB substrate produces a colorimetric signal proportional to the RASSF6 concentration. The absorbance is read at 450 nm.
Important: This kit is validated exclusively for mouse samples. It does not recognize rat or human RASSF6.
Sample Collection & Preparation
Liquid Samples
| Sample Type | Procedure |
|---|---|
| Serum | Collect in pyrogen/endotoxin-free tubes. Let blood clot, centrifuge at 3000 rpm for 10 min. Separate serum immediately. |
| Plasma | Use EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 min; collect supernatant. |
| Cell Culture Supernatant | Centrifuge at 3000 rpm for 10 min to remove cells and debris. |
Tissue Homogenates – Critical for RASSF6 Stability
RASSF6 is susceptible to endogenous proteases. To ensure accurate quantification:
- Protease Inhibitors: Freshly add a protease inhibitor cocktail (e.g., 1 mM PMSF or commercial cocktail) to the ice-cold homogenization buffer before use.
- Recommended Buffers: Standard RIPA buffer or 1× PBS (pH 7.0–7.2) containing a mild non-ionic detergent (e.g., Triton X-100, NP-40) effectively extracts native RASSF6. Keep all steps on ice.
- Homogenization: Mince tissue, add buffer at a typical ratio of 1:9 (w/v), homogenize on ice.
- Centrifugation: Centrifuge at 3000 rpm for 10 min at 4°C. Carefully collect the clear supernatant for testing.
Cell Lysates
For adherent or suspension cells, wash with cold PBS, then lyse with RIPA buffer or PBS containing 1% Triton X-100 plus protease inhibitors. Incubate on ice for 20–30 min, vortex periodically, and centrifuge at 12,000 × g for 10 min at 4°C. Use the supernatant for assay.
Storage
Aliquot and store at -20°C if not tested immediately. Avoid repeated freeze-thaw cycles. Thaw completely at room temperature and mix gently.
Critical Dilution Factor
The assay applies a 5-fold dilution directly in the well (10 µL sample + 40 µL Sample Diluent). The concentration read from the standard curve represents the diluted sample. Multiply the obtained value by 5 to obtain the actual RASSF6 concentration in the original specimen.
The S0 standard (0 pg/mL) serves as the blank/negative control.
Endogenous Level Note: RASSF6 is an intracellular protein with variable basal levels across tissues and physiological states. Serum/plasma levels are typically in the pg/mL range. We strongly recommend running a pilot study with a gradient dilution of your specific samples to ensure readings fall within the 1.0–200 pg/mL quantitative range.
Technical Specifications
| Parameter | Specification |
|---|---|
| Standard Concentrations (S0–S5) | 0, 12.5, 25, 50, 100, 200 pg/mL |
| Quantitative Range | 1.0 – 200 pg/mL |
| Analytical Sensitivity | < 1.0 pg/mL |
| Accuracy (Linearity) | Standard curve R ≥ 0.9900 |
| Precision | Intra-assay CV < 15%; Inter-assay CV < 15% |
| Specificity | Highly specific for mouse RASSF6. No cross-reactivity with mouse RASSF1, RASSF2, RASSF3, RASSF4, RASSF5, or Ras superfamily proteins (Ras, Rap). No reactivity with other soluble structural analogues. |
| Species Restriction | Validated exclusively for mouse. Not suitable for rat or human RASSF6. |
| Sample Dilution Factor | 5× (automatically performed in the well) |
| Shelf Life | 6 months |
| Storage & Transport | 2–8°C, protected from light and moisture; cold-chain shipping |
Representative Standard Curve
For spike-recovery and dilution linearity data, please refer to the product instruction manual or contact technical support.
Kit Components
96-Well Format
| Component | Quantity | Notes |
|---|---|---|
| Microelisa Stripplate | 12 strips × 8 wells | Pre-coated with anti-mouse RASSF6 antibody |
| Standard (0.3 mL/vial) | 6 vials | Ready-to-use liquid, 0–200 pg/mL |
| Sample Diluent | 6 mL | – |
| HRP-Conjugated Detection Antibody | 10 mL | Ready-to-use |
| 20× Wash Buffer | 25 mL | Dilute to 1× before use |
| Substrate A | 6 mL | – |
| Substrate B | 6 mL | – |
| Stop Solution | 6 mL | – |
| Plate Sealers | 2 sheets | – |
| Instruction Manual | 1 copy | – |
| Self-sealing Bag | 1 piece | For storing unused strips |
48-Well Format
| Component | Quantity | Notes |
|---|---|---|
| Microelisa Stripplate | 12 strips × 4 wells | Pre-coated, removable |
| Standard (0.3 mL/vial) | 6 vials | Ready-to-use liquid |
| Sample Diluent | 3 mL | – |
| HRP-Conjugated Detection Antibody | 5 mL | Ready-to-use |
| 20× Wash Buffer | 15 mL | Dilute to 1× |
| Substrate A | 3 mL | – |
| Substrate B | 3 mL | – |
| Stop Solution | 3 mL | – |
| Plate Sealers | 2 sheets | – |
| Instruction Manual | 1 copy | – |
| Self-sealing Bag | 1 piece | – |
Note: Crystallization of 20× Wash Buffer is normal; warm in a water bath until crystals dissolve.
Assay Procedure Summary
- Equilibrate: Let the foil pouch reach room temperature for 20 min. Remove required strips; seal unused strips at 4°C.
- Add Standards & Samples:
- Standard wells: 50 µL of each standard.
- Sample wells: 10 µL sample + 40 µL Sample Diluent.
- Blank well(s): Leave empty.
- Add Detection Antibody: Dispense 100 µL HRP-conjugated detection antibody to all wells except blank. Cover and incubate at 37°C for 60 minutes.
- Wash: Aspirate and blot. Fill wells with 1× Wash Buffer, let stand 1 min, discard. Repeat 5 times (automated washer: 350 µL/well, 1-min soak).
- Substrate: Add 50 µL each of Substrate A and Substrate B. Incubate in the dark at 37°C for 15 minutes.
- Stop & Read: Add 50 µL Stop Solution. Read absorbance at 450 nm within 15 minutes.
Total assay time: approximately 2 hours.
Materials Required but Not Supplied:
- Microplate reader (450 nm)
- Adjustable pipettes and tips (0.5–1000 µL)
- 37°C incubator or water bath
- Distilled water
- Absorbent paper, wash bottle or automated washer
- Protease inhibitors, RIPA buffer or detergents (for tissue/cell extraction)
Throughput
| Format | Max. Number of Samples (Single Wells) |
|---|---|
| 48-well | 39–40 samples |
| 96-well | 85–88 samples |
Includes 6 standard wells and 1–2 blank wells. For duplicates, divide accordingly.
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Frequently Asked Questions
Q: Why must I multiply the result by 5?
A: The assay dilutes sample 5-fold (10 µL sample + 40 µL diluent) in the well. The interpolated concentration corresponds to the diluted sample. Multiply by 5 to get the actual value.
Q: Does this kit cross-react with other RASSF proteins?
A: No. The kit is highly specific for mouse RASSF6 and does not recognize RASSF1, 2, 3, 4, 5 or other Ras superfamily members.
Q: Can I measure RASSF6 in rat or human samples?
A: No. The kit is validated only for mouse RASSF6. Cross-reactivity with rat or human orthologs has not been established.
Q: What buffer should I use for tissue extraction?
A: RIPA buffer or PBS with 1% Triton X-100/NP-40 is recommended. Always add fresh protease inhibitors and keep samples on ice.
Q: What are typical endogenous RASSF6 levels?
A: RASSF6 is intracellular; serum levels are low (pg/mL range). Because concentrations vary widely by tissue and disease model, we strongly recommend conducting a pilot dilution experiment for your specific samples.
Q: My reading exceeds 200 pg/mL after the 5× correction. What next?
A: Pre-dilute your sample further with Sample Diluent (e.g., 1:2 or 1:5), re-assay, and multiply by the 5× in-well dilution plus the additional dilution factor.
Ordering & Contact Information
| Item | Details |
|---|---|
| Catalog Number | Yanda-MOUSE-RASSF6 |
| Formats | 48-well / 96-well |
| Package Dimensions | 16 cm × 12.5 cm × 9.5 cm (neutral white box) |
| Shipping | Cold-chain transport (2–8°C), protected from light and moisture |
| Storage | 2–8°C; do not freeze |
| Shelf Life | 6 months |
| Intended Use | For research use only, not for diagnostic procedures |
Contact us
- WhatsApp: +86 198 3186 0669
- Email: songhawyble@gmail.com
For pricing, bulk orders, or customization, please contact us directly.
Technical Documents & Support
- Instruction Manual – Download [insert link] or available upon request.
- MSDS – Supplied with the kit or upon request.
- Technical Support – For validation data or protocol assistance, reach out via WhatsApp or email.
Disclaimer
This product is for laboratory research use only and is not approved for clinical diagnosis, treatment, or prognosis. The manufacturer assumes no responsibility for consequences arising from improper use, protocol deviations, or unauthorized applications. Users are responsible for validating assay performance in their own sample matrices and experimental conditions.
