RASSF2 Mouse mAb Protocol: WB, IHC & IP Applications

RASSF2 Mouse mAb is a monoclonal antibody raised in mouse targeting human RASSF2 protein, suitable for use in western blotting, immunohistochemistry, and immunoprecipitation applications when reconstituted and used according to the protocol below.

Product SKU

Ab351050

Clone / Reference

KC-1966

Host Species

Mouse

Target Protein

RASSF2 (Ras-association domain family member 2)

Formulation

PBS only

Antibody Class

Monoclonal antibody (mAb)

Overview of RASSF2 and Antibody Specifications

RASSF2 (Ras-association domain family member 2) is a member of the RASSF tumor suppressor family. It acts as a negative regulator of Ras-driven cell proliferation and has been implicated in the regulation of apoptosis and cell cycle progression. Epigenetic silencing of RASSF2 has been observed in multiple human cancers, making it a biologically relevant target for oncology research.

The RASSF2 Mouse mAb (Ab351050, clone KC-1966) is a monoclonal antibody produced in mouse and formulated in PBS. The PBS-only formulation means no carrier proteins or preservatives are present, which is advantageous for applications requiring low background or when the antibody will be directly conjugated. Researchers should add appropriate preservatives (e.g., sodium azide to 0.02% final concentration) and a carrier protein (e.g., BSA to 0.1%) if long-term storage is required.

Sample Preparation

Cell and Tissue Lysates (for Western Blot and IP):

1. Harvest cells by scraping or centrifugation. Wash twice with ice-cold PBS.
2. Lyse cells in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail. Incubate on ice for 30 minutes with occasional vortexing.
3. Centrifuge at 12,000 × g for 15 minutes at 4 °C. Collect the supernatant.
4. Quantify protein concentration using a BCA or Bradford assay.
5. For tissue samples, homogenize fresh or frozen tissue in lysis buffer using a mechanical homogenizer before proceeding with steps 2–4.

Tissue Sections (for IHC):

1. Fix tissue in 10% neutral-buffered formalin and embed in paraffin (FFPE) or prepare frozen sections.
2. Cut sections at 4–6 µm thickness and mount on positively charged slides.
3. For FFPE sections, deparaffinize in xylene and rehydrate through a graded ethanol series to distilled water.
4. Perform antigen retrieval by heating sections in citrate buffer (10 mM sodium citrate, pH 6.0) or EDTA buffer (1 mM EDTA, pH 9.0) at 95–100 °C for 10–20 minutes.

Recommended Protocol by Application

Western Blot (WB):

1. Separate 20–40 µg of total protein per lane by SDS-PAGE using an appropriate percentage gel based on the molecular weight of RASSF2 (~36 kDa).
2. Transfer proteins to a PVDF or nitrocellulose membrane.
3. Block the membrane in 5% non-fat dry milk or 3% BSA in TBST for 1 hour at room temperature.
4. Incubate with RASSF2 Mouse mAb (Ab351050) at the manufacturer's recommended dilution in blocking buffer overnight at 4 °C with gentle rocking. (Consult the datasheet for the validated working dilution range; see review flag.)
5. Wash the membrane 3 × 10 minutes with TBST.
6. Incubate with an appropriate HRP-conjugated anti-mouse secondary antibody for 1 hour at room temperature.
7. Wash 3 × 10 minutes with TBST and detect signal using an ECL substrate.

Immunohistochemistry (IHC):

1. After antigen retrieval, allow slides to cool to room temperature and rinse with PBS.
2. Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 minutes.
3. Block non-specific binding with 5–10% normal serum (from the secondary antibody host species) for 30 minutes.
4. Incubate sections with RASSF2 Mouse mAb (Ab351050) at the recommended dilution overnight at 4 °C in a humidified chamber. (Consult the datasheet for validated IHC dilution; see review flag.)
5. Wash 3 × 5 minutes in PBS. Apply appropriate biotinylated secondary antibody or polymer detection system for 30–60 minutes.
6. Develop with DAB substrate, counterstain with hematoxylin, dehydrate, and mount.

Immunoprecipitation (IP):

1. Pre-clear 500 µg cell lysate with Protein G or Protein A agarose beads for 1 hour at 4 °C with rotation.
2. Add RASSF2 Mouse mAb (Ab351050) at the manufacturer's recommended amount to the pre-cleared lysate. Rotate overnight at 4 °C. (Consult the datasheet for validated IP antibody amount; see review flag.)
3. Add fresh Protein G or Protein A agarose beads and incubate for 2 hours at 4 °C with rotation.
4. Wash beads 4–5 times with lysis buffer.
5. Elute bound proteins by boiling in 2× SDS sample buffer for 5 minutes.
6. Resolve eluate by SDS-PAGE and proceed with western blot detection as above.

Storage, Handling and Quality Notes

Storage: Store RASSF2 Mouse mAb (Ab351050) at –20 °C for long-term storage. For short-term use (up to 1 month), aliquots may be stored at 4 °C. Avoid repeated freeze-thaw cycles, which can denature the antibody and reduce sensitivity. Prepare single-use aliquots upon first thaw.

Formulation note: This antibody is supplied in PBS only (no stabilizing proteins or preservatives). If storing for extended periods, consider supplementing with BSA (0.1% final) and sodium azide (0.02% final). Note that sodium azide is toxic — handle with appropriate precautions and do not use azide-containing preparations in peroxidase-based detection systems, as azide inhibits HRP activity.

Sterility and contamination: Work under clean conditions and use aseptic technique when aliquoting. Microbial contamination will degrade antibody performance.

Quality control: Each lot is manufactured under controlled conditions. Verify lot-specific performance data on the product datasheet available from Aladdin Scientific (Ab351050).

Troubleshooting

Problem	Possible Cause	Suggested Solution
No signal (WB)	Antibody dilution too high; insufficient antigen; transfer failure	Titrate antibody concentration; increase protein load; verify transfer efficiency with Ponceau S staining
High background (WB/IHC)	Insufficient blocking; secondary antibody cross-reactivity; too much primary antibody	Increase blocking time or concentration; optimize primary antibody dilution; increase wash stringency
Multiple bands (WB)	Protein degradation; non-specific binding	Add fresh protease inhibitors during lysis; optimize antibody dilution; confirm expected RASSF2 molecular weight (~36 kDa)
Weak or no IP signal	Insufficient antibody; poor bead capture; antigen–antibody incompatibility under lysis conditions	Increase antibody amount; use fresh beads; test alternative lysis conditions with milder detergents
High background (IHC)	Endogenous peroxidase not fully quenched; inadequate antigen retrieval	Extend H₂O₂ quenching step; optimize antigen retrieval buffer and time

Frequently asked questions

What is the recommended storage condition for RASSF2 Mouse mAb (Ab351050)?

Store at –20 °C for long-term storage. For short-term use, aliquots can be kept at 4 °C for up to one month. Avoid repeated freeze-thaw cycles by preparing single-use aliquots upon first thaw.

Why is the PBS-only formulation important, and are there any special handling considerations?

The PBS-only formulation contains no carrier proteins (e.g., BSA) or preservatives (e.g., sodium azide), making it suitable for direct conjugation and minimizing assay background. However, this also means the antibody is more susceptible to degradation; researchers should add BSA and sodium azide if extended storage is needed, and handle the stock under aseptic conditions.

What applications has RASSF2 Mouse mAb been validated for?

Based on the product information, RASSF2 Mouse mAb (Ab351050, KC-1966) is suitable for use in western blotting (WB), immunohistochemistry (IHC), and immunoprecipitation (IP). Please consult the current product datasheet on the Aladdin Scientific website for the full list of validated applications and species reactivity.

What is the expected molecular weight of RASSF2 by western blot?

RASSF2 has a predicted molecular weight of approximately 36 kDa. The observed band by western blot may differ slightly due to post-translational modifications or anomalous SDS-PAGE migration. Please verify against the datasheet for lot-specific observations.

Can sodium azide affect the performance of RASSF2 Mouse mAb in HRP-based detection assays?

Yes. Sodium azide is a potent inhibitor of horseradish peroxidase (HRP). If sodium azide has been added to the antibody stock for preservation, ensure thorough dilution before use in HRP-based assays, or switch to an alternative preservation method for lots intended for these applications.

References

1. Vos MD et al. A role for the RASSF1A tumor suppressor in the regulation of tubulin polymerization and genomic stability. Cancer Research, 2000.
2. Dallol A et al. RASSF2, a candidate tumor suppressor gene at human chromosome 20p13. Cancer Research, 2004.
3. Aladdin Scientific. RASSF2 Mouse mAb (Ab351050) Product Datasheet. Aladdin Scientific Inc.

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