RBM3 Mouse mAb: Application Guide & Protocol Tips

RBM3 Mouse mAb is a monoclonal antibody raised in mouse that specifically targets RNA Binding Motif Protein 3 (RBM3), a cold-inducible RNA-binding protein implicated in stress response, neuroprotection, and cancer biology.

Product SKU

Ab349618

Antibody Type

Mouse Monoclonal Antibody (mAb)

Target Protein

RBM3 (RNA Binding Motif Protein 3)

Reference Catalog

absea#KC-12540

Recommended Applications

Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)

Host Species

Mouse

Overview of RBM3 and Its Biological Significance

RBM3 (RNA Binding Motif Protein 3) is a member of the glycine-rich RNA-binding protein family and contains a single RNA recognition motif (RRM). It is encoded by a gene located on the X chromosome and is classically induced by mild hypothermia and hypoxia, earning it the designation of a cold-shock protein.

RBM3 plays critical roles in global protein synthesis regulation, particularly under stress conditions. It has been identified as a cytoprotective factor in neuronal cells, with studies suggesting its upregulation can delay synaptic loss and neurodegeneration. Additionally, RBM3 expression has been reported to be dysregulated in several human cancers, making it a relevant biomarker for oncology research.

The RBM3 Mouse mAb (Ab349618) provides researchers with a high-specificity tool to detect and study endogenous RBM3 protein across a range of experimental platforms.

Recommended Protocols by Application

Western Blot (WB)

1. Lyse cells or tissue in RIPA buffer supplemented with protease inhibitors. Determine protein concentration by BCA or Bradford assay.

2. Load 20–40 µg of total protein per lane on a 12–15% SDS-PAGE gel. Transfer to PVDF or nitrocellulose membrane.

3. Block membrane with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature.

4. Incubate with RBM3 Mouse mAb at the manufacturer-recommended dilution overnight at 4°C with gentle agitation.

5. Wash 3× with TBST (10 minutes each). Apply appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.

6. Detect with ECL reagent. Expected band size for RBM3 is approximately 17 kDa.

Immunohistochemistry (IHC)

1. Fix tissue in 10% neutral buffered formalin and embed in paraffin (FFPE). Cut 4–5 µm sections and mount on positively charged slides.

2. Deparaffinize in xylene and rehydrate through a graded ethanol series to water.

3. Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) by heat-induced epitope retrieval (HIER).

4. Block endogenous peroxidase with 3% H₂O₂ for 10 minutes. Apply protein block for 30 minutes.

5. Incubate with RBM3 Mouse mAb at appropriate dilution overnight at 4°C. Apply secondary antibody and DAB chromogen. Counterstain with hematoxylin.

Immunofluorescence (IF)

1. Seed cells on coverslips. Fix with 4% paraformaldehyde for 15 minutes and permeabilize with 0.1–0.3% Triton X-100 for 10 minutes.

2. Block with 5% normal serum for 1 hour. Incubate with RBM3 Mouse mAb overnight at 4°C.

3. Apply fluorophore-conjugated anti-mouse secondary antibody for 1 hour. Counterstain nuclei with DAPI. Mount coverslips and image by fluorescence microscopy.

Optimization and Troubleshooting

High Background: Increase blocking time or concentration. Ensure thorough washing steps between antibody incubations. Titrate primary antibody to the optimal working dilution using a serial dilution experiment.

No Signal or Weak Signal: Verify sample expression of RBM3 — consider using a positive control cell line known to express RBM3 (e.g., cells exposed to mild cold shock at 32°C). Confirm that secondary antibody is compatible with a mouse primary antibody host. Check protein transfer efficiency.

Non-Specific Bands (WB): Lower primary antibody concentration. Increase stringency of washes (add 0.05–0.1% Tween-20 to wash buffer). Run a negative control lane with isotype control antibody.

Storage and Handling: Store the antibody at –20°C for long-term storage. Avoid repeated freeze-thaw cycles; aliquot upon receipt. Do not use antibody that has been left at room temperature for extended periods.

Controls and Validation

Rigorous experimental controls are essential for interpreting results obtained with RBM3 Mouse mAb:

• Positive Control: Use lysate or tissue from a source confirmed to express RBM3, such as brain tissue or cells treated with mild hypothermia (32°C, 24 hours).
• Negative Control: Replace primary antibody with an isotype-matched mouse IgG at equivalent concentration.
• Loading Control (WB): Probe with a housekeeping protein antibody (e.g., anti-β-actin or anti-GAPDH) to confirm equal protein loading.
• Knockout Validation: Where possible, use RBM3 knockout cell lines or siRNA knockdown samples to confirm band or signal specificity.

Validation across multiple applications (WB + IHC or WB + IF) increases confidence in the antibody's specificity for your experimental system.

Sample Preparation Considerations

RBM3 is primarily a nuclear protein with some cytoplasmic distribution depending on cell type and stress conditions. When preparing cell lysates, use complete lysis buffers to ensure efficient extraction of nuclear proteins. Nuclear fractionation may be employed if subcellular localization studies are intended.

For tissue samples, rapid processing and fixation are important to preserve RBM3 epitopes. Prolonged formalin fixation can reduce antibody accessibility; optimize fixation time according to tissue type.

When studying cold-shock induction of RBM3, culture cells at 32°C for 24–48 hours as a standard mild hypothermia protocol. Include parallel normothermia controls (37°C) to demonstrate induction. Whole-cell lysate prepared immediately after treatment will best reflect RBM3 expression levels.

Frequently asked questions

What is the expected molecular weight of RBM3 detected by this antibody?

RBM3 has a predicted molecular weight of approximately 17 kDa. In Western blot experiments, the band may appear slightly higher or lower depending on the gel percentage and running conditions. Confirm with positive control lysate.

Which species does the RBM3 Mouse mAb (Ab349618) recognize?

The species reactivity of Ab349618 should be confirmed with the official product datasheet from Aladdin Scientific. Commonly, antibodies targeting RBM3 may cross-react with human and mouse RBM3 due to high sequence conservation, but please verify reactivity data before use.

How should I store the RBM3 Mouse mAb to maintain its activity?

Store the antibody at –20°C for long-term use. Avoid repeated freeze-thaw cycles by preparing single-use aliquots upon receipt. For short-term use within one to two weeks, storage at 4°C is acceptable. Refer to the product datasheet for the exact formulation and storage recommendations.

Can I use RBM3 Mouse mAb for flow cytometry?

Flow cytometry application has not been confirmed in the current source material for Ab349618. Please consult the official product datasheet or contact Aladdin Scientific technical support to verify whether flow cytometry is a validated application for this antibody.

What positive control sample is recommended for RBM3 detection?

Cells exposed to mild hypothermia (32°C for 24–48 hours) serve as a reliable positive control due to strong transcriptional induction of RBM3 under cold-shock conditions. Brain tissue lysate is also widely reported to express RBM3 at detectable levels.

References

1. Chip S, Zelmer A, Ogunshola OO, et al. The RNA-binding protein RBM3 is involved in hypothermia induced neuroprotection. Neurobiology of Disease. 2011.
2. Wellmann S, Buhrer C, Moderegger E, et al. Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism. Journal of Cell Science. 2004.
3. Zeng Y, Kulkarni P, Irimia-Dominguez J, Bhatt DL. RBM3 expression in cancer: a review. 2023.

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