Recombinant RRM2B Antibody: Usage & Protocol Guidelines

Recombinant RRM2B Antibody is a research-grade reagent designed to detect and characterize the RRM2B (p53R2) protein in a variety of immunoassay applications, supporting studies of DNA repair, mitochondrial function, and p53-dependent stress responses.

Product SKU

Ab350697

Antibody Type

Recombinant antibody

Formulation

PBS only (absea#RN-049)

Target Protein

RRM2B (p53R2, Ribonucleotide Reductase M2B)

Recommended Applications

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

Storage Buffer

PBS (Phosphate-Buffered Saline)

Background: RRM2B Biology and Research Significance

RRM2B, also known as p53R2, encodes the small subunit of ribonucleotide reductase (RNR) that is transcriptionally induced by the tumor suppressor p53. Unlike the cell-cycle-regulated RRM2 subunit, RRM2B is expressed in non-dividing, differentiated cells and plays a critical role in supplying deoxyribonucleotide precursors for mitochondrial DNA (mtDNA) synthesis and repair. Loss-of-function mutations in RRM2B have been associated with mtDNA depletion syndromes, progressive external ophthalmoplegia, and other mitochondrial disorders. In the context of the DNA damage response, RRM2B supports nucleotide pool replenishment required for p53-dependent DNA repair, making it a relevant target in cancer biology, aging research, and mitochondrial disease studies.

Reagent Specifications and Storage Guidelines

The Recombinant RRM2B Antibody (Ab350697) is supplied in PBS (phosphate-buffered saline) only, without carrier proteins or preservatives (catalog reference: absea#RN-049). Key handling guidelines are as follows:

• Storage temperature: Aliquot upon receipt and store at −20°C for long-term use. Avoid repeated freeze-thaw cycles, which can reduce antibody activity.
• Short-term storage: If the antibody will be used within 1–2 weeks, store at 4°C.
• Formulation note: Because the PBS-only formulation lacks stabilizing proteins such as BSA or glycerol, consider adding a carrier protein (e.g., 0.1% BSA) or glycerol (50% final concentration) if long-term stability is required, and re-aliquot accordingly.
• Sterility: Handle under aseptic conditions where possible to prevent microbial contamination of the PBS-based solution.

Always centrifuge the vial briefly before opening to recover any antibody from the cap or walls of the tube.

Step-by-Step Application Protocols

Western Blot (WB)

1. Lyse cells or tissues in RIPA buffer supplemented with protease inhibitors. Determine protein concentration by BCA or Bradford assay.
2. Separate 20–40 μg total protein by SDS-PAGE (typically 10–12% gel for RRM2B, expected MW ~40 kDa) and transfer to PVDF or nitrocellulose membrane.
3. Block membrane with 5% non-fat milk or 5% BSA in TBST for 1 hour at room temperature.
4. Incubate with Recombinant RRM2B Antibody (Ab350697) at the recommended dilution (refer to product datasheet; typical starting range 1:500–1:2000) in blocking buffer overnight at 4°C.
5. Wash 3× with TBST (10 minutes each), then incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature.
6. Detect signal by ECL chemiluminescence and image on X-ray film or digital imager.

Immunohistochemistry (IHC-P)

1. Deparaffinize and rehydrate FFPE sections through a standard xylene/ethanol series.
2. Perform antigen retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) using a pressure cooker or microwave method.
3. Block endogenous peroxidase with 3% H₂O₂ for 10 minutes, then block with 5% normal serum for 30 minutes.
4. Apply Recombinant RRM2B Antibody (Ab350697) at the recommended dilution and incubate overnight at 4°C in a humidified chamber.
5. Apply appropriate biotinylated secondary antibody, then ABC reagent; develop with DAB substrate and counterstain with hematoxylin.

Immunofluorescence (IF)

1. Fix cells with 4% paraformaldehyde (10 minutes, RT) and permeabilize with 0.1–0.3% Triton X-100 in PBS.
2. Block with 5% normal serum in PBS for 30–60 minutes at RT.
3. Incubate with Recombinant RRM2B Antibody (Ab350697) at the recommended dilution overnight at 4°C.
4. Wash 3× with PBS, then incubate with fluorophore-conjugated secondary antibody for 1 hour at RT in the dark.
5. Mount with DAPI-containing mounting medium and image by fluorescence microscopy.

Troubleshooting Common Issues

The table below summarizes the most frequently encountered problems when using Recombinant RRM2B Antibody (Ab350697) and suggested remedies:

• No signal / weak signal: Verify that the target protein is expressed in your cell or tissue model. Increase antibody concentration or extend primary antibody incubation time. Ensure antigen retrieval is optimized for IHC. Check that secondary antibody is compatible with the host species of the primary antibody.
• High background: Increase blocking time or switch blocking agent (e.g., from milk to BSA for phospho-targets). Increase number or duration of wash steps. Reduce primary antibody concentration. Ensure secondary antibody dilution is appropriate.
• Multiple non-specific bands on WB: Confirm protein was not degraded during lysis (use fresh protease inhibitors). Try reducing SDS-PAGE running time or adjusting transfer conditions. Consider optimizing the antibody dilution.
• Inconsistent IHC staining: Standardize antigen retrieval method and duration. Verify tissue fixation time was consistent (over-fixation can mask epitopes). Use a validated positive control tissue known to express RRM2B.
• Antibody precipitation: This may occur if the PBS-only formulation was stored improperly. Centrifuge at low speed before use and ensure the antibody was not subjected to repeated freeze-thaw cycles.

Frequently asked questions

What applications is Recombinant RRM2B Antibody (Ab350697) validated for?

The Recombinant RRM2B Antibody (Ab350697) is intended for use in standard immunoassay applications including Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF). Always confirm validated applications and recommended dilutions on the product datasheet, as performance can vary by application and sample type.

Why is the antibody formulated in PBS only, and how does this affect storage?

The PBS-only formulation (absea#RN-049) keeps the antibody free from carrier proteins or preservatives that could interfere with certain downstream assays. However, the absence of stabilizers such as BSA or glycerol means the antibody may be more susceptible to loss of activity during storage. It is strongly recommended to aliquot the antibody upon receipt and store aliquots at -20°C to minimize freeze-thaw cycles and maintain activity.

What positive control samples can be used to validate RRM2B antibody performance?

RRM2B (p53R2) is expressed in a variety of human tissues, with notable expression reported in tissues with high metabolic and DNA repair demands. Cell lines with confirmed RRM2B expression or tissues known to be RRM2B-positive (as referenced in validated databases such as the Human Protein Atlas) can serve as positive controls. Please consult the product datasheet for Aladdin-recommended positive control samples specific to Ab350697.

Can the Recombinant RRM2B Antibody be used for immunoprecipitation (IP)?

Immunoprecipitation compatibility is not confirmed in the available source material for Ab350697. Contact Aladdin Scientific technical support for the latest application validation data before attempting IP experiments with this antibody.

References

1. Tanaka H, Arakawa H, Yamaguchi T, et al. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature. 2000;404(6773):42-49. doi:10.1038/35003506
2. Bourdon A, Minai L, Serre V, et al. Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nature Genetics. 2007;39(6):776-780. doi:10.1038/ng2040
3. Aladdin Scientific. Recombinant RRM2B Antibody (Ab350697) Product Datasheet. Aladdin Scientific; current edition.

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