ORRM2 Antibody

What is RRM2 and what is its biological function?

RRM2 is one of two non-identical subunits (alongside RRM1) that constitute ribonucleotide reductase. This enzyme catalyzes the conversion of ribonucleotides to deoxyribonucleotides, which is a rate-limiting step in the production of dNTPs required for DNA synthesis and repair .

RRM2's biological functions include:

• DNA synthesis during cell replication

• Cell cycle regulation (expressed during late G1/early S phase, degraded in late S phase)

• Potential role in ferroptosis inhibition

• Wnt signaling inhibition

The activity of ribonucleotide reductase, and consequently DNA synthesis and cell proliferation, is controlled during the cell cycle by the synthesis and degradation of the RRM2 subunit .

What applications are most commonly validated for RRM2 antibodies?

Based on commercial antibody validation data, RRM2 antibodies have been successfully used in multiple applications:

Most antibodies demonstrate strong performance in WB applications, with recommended dilutions typically ranging from 1:1000 to 1:50000 depending on the specific antibody clone .

What is the typical molecular weight observed for RRM2 in western blotting?

The calculated molecular weight of RRM2 is approximately 45 kDa, which is consistently observed across western blotting applications using various antibodies . This consistency in observed molecular weight provides a useful validation parameter when testing new RRM2 antibodies in your experimental system.

In which tissues and cell types is RRM2 typically expressed?

According to published literature and manufacturer data, RRM2 expression has been detected in:

Normal tissues:

• Secondary oocyte

• Neonatal skin

• Muscle and skin

• Stomach

Cancer tissues/cell lines:

• Mammary carcinoma

• Cervix carcinoma

• Leukemic T-cells

• Colon carcinoma

• Liver hepatocellular carcinoma (HepG2 cells)

• Multiple cancer types according to UALCAN database (including BLCA, BRCA, COAD, etc.)

Western blot detection has been validated in multiple cell lines including HeLa, HEK-293, MCF-7, A431, Jurkat, K-562, and THP-1 cells .

What are the optimal conditions for immunohistochemistry with RRM2 antibodies?

Based on protocols provided by antibody manufacturers:

Antigen Retrieval:

• Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) is most commonly recommended

• Alternative: citrate buffer pH 6.0 may be used for some tissue types

Incubation Conditions:

• Typical dilution range: 1:1000-1:8000 (depending on antibody)

• Recommended incubation time: 30 minutes at room temperature

Detection Systems:

• Polymer-based detection systems like LeicaDS9800 (Bond® Polymer Refine Detection) have shown good results

Counterstain:

• Hematoxylin is the standard counterstain used in validated protocols

Notable Considerations:

• Negative/low expression control: human cerebrum has shown minimal RRM2 staining

• Positive controls: human colon carcinoma and stomach tissues have shown consistent positive staining

How should researchers optimize western blotting protocols for RRM2 detection?

For optimal western blotting results with RRM2 antibodies:

Sample Preparation:

• Cell lysates from HeLa, HEK-293, HepG2, and other cancer cell lines provide reliable positive controls

Electrophoresis Conditions:

• Standard 10% SDS-PAGE gels are suitable for resolving the 45 kDa RRM2 protein

Transfer and Blocking:

• Follow standard protein transfer protocols

• Blocking with normal goat serum (10%) combined with 0.3M glycine has been reported for flow cytometry applications and may be applicable to western blotting

Antibody Dilutions:

• Primary antibody: Ranges from 1:1000 to 1:50000 depending on specific antibody clone

• For example, ab57653 (clone 1E1) has been used at 1 μg/mL

• For higher sensitivity applications, recombinant monoclonal antibodies like EPR11820 may offer advantages

Detection:

• Standard HRP-conjugated secondary antibodies with ECL detection systems are suitable

What are the validated protocols for immunofluorescence with RRM2 antibodies?

For immunofluorescence applications:

Cell Fixation Methods:

• Methanol fixation has been validated for A549 cells

• For HeLa cells, standard PFA fixation followed by permeabilization has been used

Dilution Range:

• Typical range: 1:100-1:800 depending on the antibody clone

• For polyclonal antibodies like ab154964, 1:500 dilution has shown good results

Counterstaining:

• Nuclear counterstaining with DAPI or Hoechst 33342 is recommended

Validated Cell Lines:

• HeLa, A549, and HepG2 cells have been successfully used for IF/ICC applications

How can RRM2 antibodies be used to investigate cancer biomarkers and prognosis?

RRM2 has emerged as a significant biomarker in multiple cancer types with important clinical implications:

Breast Cancer:

• RRM2 expression (detected by IHC) correlates with molecular subtypes, with higher expression in non-luminal cases

• In ER-positive breast cancers, RRM2 expression associates with shorter disease-free survival (borderline significance)

• High RRM2 scores are more common in triple-negative breast cancer and HER2-enriched subtypes (71.8% and 86.2% respectively)

Liver Cancer:

• Serum RRM2 levels (detected by ELISA) show diagnostic potential for liver cancer

• RRM2 serum concentration correlates with AFP (R=0.45, P<0.0001) and CEA (R=0.45, P<0.0001)

• ROC analysis shows RRM2 has better diagnostic performance (AUC: 0.863, 95% CI 0.821–0.904) than AFP (AUC: 0.798, 95% CI 0.745–0.851)

• Combining RRM2 with AFP improves diagnostic performance (AUC: 0.947, sensitivity 88.7%, specificity 97.0%)

Pan-Cancer Analysis:

What is the relationship between RRM2 and ferroptosis in cancer research?

RRM2 has emerged as a key protector against ferroptosis, particularly in liver cancer:

Mechanism of Action:

• Inhibition of glutathione (GSH) synthesis leads to ferroptosis

• RRM2 protects against ferroptosis, and this protection is linked to liver cancer progression

• Knockdown of RRM2 significantly increases susceptibility to ferroptosis

Clinical Implications:

• RRM2 serum levels may serve as a biomarker not only for cancer diagnosis but also for predicting ferroptosis resistance

• This could help identify patients who might benefit from ferroptosis-based treatments

Research Applications:

• RRM2 antibodies can be used to study ferroptosis mechanisms in cancer cells

• Immunoblotting or IHC for RRM2 in cancer tissues may help predict response to ferroptosis-inducing therapies

• Combined with other ferroptosis markers, RRM2 detection could provide a more comprehensive understanding of ferroptosis resistance in tumors

How does RRM2 expression correlate with immune infiltration in cancer?

Recent pan-cancer analyses have revealed important relationships between RRM2 and immune parameters:

Correlation with Immune Cells:

• RRM2 expression significantly associates with Natural Killer T (NKT) cell infiltration

• Positive correlation with immune scores in several cancer types

Immunotherapy Implications:

• RRM2 levels positively correlate with:

  • Immune checkpoint expression

  • Tumor mutation burden

  • Microsatellite instability

  • Neoantigen presence

  • Cytotoxic T lymphocyte infiltration

Research Applications:

• Using RRM2 antibodies in multiplex IHC alongside immune markers may help predict immunotherapy response

• Detection of RRM2 in patient samples could potentially identify candidates who would benefit from immunotherapy

• Combined RRM2/immune checkpoint analysis may offer improved predictive value

How can researchers validate the specificity of RRM2 antibodies?

To ensure antibody specificity:

Positive Controls:

• Use established cell lines with confirmed RRM2 expression:

  • HeLa cells for western blot

  • HepG2 cells for IF/ICC and western blot

  • Human colon carcinoma for IHC

Negative Controls:

• Human cerebrum tissue shows minimal RRM2 expression (suitable negative control for IHC)

• Use appropriate isotype controls for all applications

• When possible, include RRM2 knockdown samples as specificity controls

Expected Results:

• Western blot: Single band at approximately 45 kDa

• IHC/IF: Predominantly cytoplasmic staining pattern

• The staining intensity should correlate with cell proliferation status (higher in actively dividing cells)

Cross-Reactivity Assessment:

• While most antibodies are validated for human RRM2, some may cross-react with other species

• Check sequence homology before using human RRM2 antibodies on non-human samples

What are common challenges and solutions when using RRM2 antibodies?

How do monoclonal and polyclonal RRM2 antibodies compare in performance?

Monoclonal Antibodies:

• Clones available include 1E1 , EPR11820

• Advantages:

  • Consistent lot-to-lot reproducibility

  • High specificity for single epitope

  • Lower background in most applications

• Best applications:

  • Western blot (1E1 clone has been widely cited)

  • Flow cytometry (validated for clone 1E1)

  • Quantitative applications requiring high specificity

Polyclonal Antibodies:

• Available from multiple vendors including Proteintech (11661-1-AP)

• Advantages:

  • Recognition of multiple epitopes

  • Often higher sensitivity for low-abundance targets

  • May be more robust to protein denaturation/modifications

• Best applications:

  • IHC on formalin-fixed tissues

  • IP applications (cited in publications)

  • Detection of proteins with post-translational modifications

Recombinant Antibodies:

• Example: EPR11820 (ab172476)

• Advantages:

  • Combines specificity of monoclonals with consistent reproducibility

  • No batch-to-batch variation

  • Often higher affinity and specificity

• Ideal for:

  • Critical research requiring highest reproducibility

  • Multiple applications (validated for WB, IP, IHC-P, ICC/IF)

How can RRM2 antibodies be used to study cell cycle regulation?

RRM2 expression is tightly regulated during the cell cycle:

• Only expressed during late G1/early S phase

• Degraded in late S phase

• Activity of ribonucleotide reductase is controlled by synthesis and degradation of RRM2

Research Applications:

• Use RRM2 antibodies in combination with cell cycle markers to study cell cycle progression

• Flow cytometry with RRM2 antibodies can help identify cells in S phase

• Monitor RRM2 expression changes after cell cycle-disrupting drugs

• Study the mechanisms of RRM2 degradation in late S phase

Experimental Approach:

• Synchronize cells at different cell cycle stages

• Perform western blot or flow cytometry with RRM2 antibodies

• Correlate RRM2 expression with established cell cycle markers

• Use RRM2 knockdown to study effects on cell cycle progression (significant decrease in proliferation during S phase has been reported)

What insights can RRM2 detection provide about drug resistance mechanisms?

RRM2 has been implicated in drug resistance mechanisms, particularly for tamoxifen in breast cancer:

Tamoxifen Resistance:

• RRM2 is involved in tamoxifen resistance in breast cancer by regulating cell growth and DNA damage via protein kinase B (AKT) pathway reversal

• Inhibition of RRM2 can reverse tamoxifen resistance in vivo and reduce invasive potential in vitro

Research Applications:

• Use RRM2 antibodies to monitor expression changes in drug-resistant vs. sensitive cells

• Correlate RRM2 expression with treatment response in patient samples

• Study the relationship between RRM2 and AKT pathway activation in resistant cells

• Investigate the potential of RRM2 inhibitors as adjuvant therapy in resistant tumors

Experimental Design:

• Develop drug-resistant cell lines through continuous exposure

• Compare RRM2 expression between parent and resistant lines using various antibody-based methods

• Perform RRM2 knockdown experiments to assess reversal of resistance

• Use antibodies for co-IP studies to identify RRM2 interaction partners in resistance mechanisms