SRRM2 Antibody

What is SRRM2 and what is its normal cellular function?

SRRM2 (also known as SRM300) is a large, mostly unstructured serine/arginine-rich (SR) protein that serves as a core scaffold protein required for proper formation of nuclear speckles. It contains an N-terminal RNA recognition motif and a large serine/arginine-rich C-terminal low-complexity intrinsically disordered region (IDR) that mediates protein-protein interaction and liquid-liquid phase separation (LLPS) . SRRM2 plays a central role in mRNA splicing and, as a member of the SR family, affects alternative splice sites both in vitro and in vivo . Interestingly, SRRM2 has been found to accumulate in neuron cytoplasm in Alzheimer's disease, frontotemporal dementia, and other neurodegenerative diseases .

How do I confirm the specificity of an SRRM2 antibody?

To validate SRRM2 antibody specificity:

• Perform immunoprecipitation with the SRRM2 antibody followed by Western blot analysis

• Use multiple antibodies targeting different epitopes of SRRM2 to confirm results

• Test against truncated versions of SRRM2 to identify binding regions (e.g., SRRM2 tr0 and SRRM2 tr10)

• Include isotype control antibodies for flow cytometry and immunoprecipitation

• Use siRNA knockdown of SRRM2 to confirm specificity through signal reduction

• Confirm expected molecular weight (~300 kDa for full-length SRRM2)

In published validation studies, near-full-length SRRM2 tr0 was recognized by mAb SC35 to the same extent as SRRM2 polyclonal antibody, while SRRM2 tr10 was not detected by mAb SC35 but was strongly detected with SRRM2 polyclonal antibody .

What cell types express SRRM2 on their surface?

SRRM2 has been detected on the surface of:

• Most cancer cell lines from various entities

• Acute myeloid leukemia (AML) blast cells, with expression levels correlating to high risk, relapse, and poor prognosis

• Multiple myeloma (MM) plasma cells, with expression rates of 57.64%, 64.38%, and 98.18% in RPMI-8226, U226, and H929 cell lines, respectively

• Patient-derived cancer cells in vivo

Importantly, in normal hematopoietic stem cells, differentiated mature blood cells, and normal tissues (e.g., lung), SRRM2 was only found to be located in the nucleus, not on the cell surface , making it a potential cancer-specific target.

What protocols are recommended for SRRM2 antibody use in Western blotting?

For optimal Western blotting with SRRM2 antibodies:

• Lyse cells in RIPA buffer with protease inhibitors

• Separate proteins using SDS-PAGE (note that SRRM2 is ~300 kDa)

• Transfer to appropriate membranes

• Block with suitable blocking buffer

• Incubate with SRRM2-specific primary antibody

• Wash in TBS/0.05% Tween-20

• Incubate with HRP-coupled secondary antibodies at room temperature for 2 hours

• Develop with ECL

• Quantify signals on an imaging system (e.g., Vilber Fusion FX6)

For immunoprecipitations, protocols using activated CNBr beads (Sepharose 4 Fast Flow, GE Healthcare) with appropriate coupling buffers have been successfully implemented .

What mechanisms lead to the ectopic expression of SRRM2 on cancer cell surfaces?

While the precise mechanisms remain under investigation, research indicates:

• SRRM2 is released from cancer cells via extracellular vesicles (EVs)

• The membrane composition of EVs largely reflects that of the plasma membrane of the cell of origin

• Aberrant protein localization in cancer cells may transport nuclear proteins to the cell surface

• Surface expression appears to be a cancer-specific phenomenon not observed in normal cells

This represents a critical area for future research, as understanding the mechanisms of SRRM2 surface exposure could provide insights into cancer biology and potential therapeutic interventions.

How does SRRM2 expression correlate with clinical outcomes in different cancer types?

SRRM2 expression has significant clinical correlations:

• In multiple myeloma:

  • High SRRM2 expression correlates with elevated serum β2-mg and LDH levels

  • Associated with higher ISS staging and plasma cell infiltration

  • Linked to high-risk mSMART 3.0 stratification

  • Correlated with cytogenetic abnormalities, particularly 1q21 amplification

  • In previously treated MM patients, associated with more relapses and fewer autologous stem cell transplant treatments

• In acute myeloid leukemia:

  • Surface SRRM2 expression correlates with high risk, relapse, and poor prognosis

  • Expression on blast cells from various types of AML cell lines and patients with AML

• Other cancers:

  • Increased SRRM2 expression is associated with poor survival in renal and hepatocellular carcinoma according to proteinatlas.org and BloodSpot databases

These correlations establish SRRM2 as a potential prognostic biomarker across multiple cancer types.

What methodologies are effective for detecting surface versus intracellular SRRM2?

To distinguish between surface and intracellular SRRM2:

• Flow cytometry:

  • For surface detection: Stain cells without permeabilization

  • For total SRRM2: Use permeabilization protocols

  • Compare results to quantify surface versus intracellular distribution

• Microscopy techniques:

  • Immunohistochemistry has successfully demonstrated high SRRM2 expression in bone marrow of MM patients with high plasma cell infiltration

  • Confocal microscopy with differential staining can distinguish locations

• Biochemical approaches:

  • Cell surface biotinylation followed by pulldown

  • Cellular fractionation to separate membrane from nuclear fractions

• Controls:

  • Use normal cells as controls (should only show nuclear SRRM2)

  • Include isotype antibody controls to ensure specificity

These methods have successfully distinguished surface SRRM2 on cancer cells from the exclusively nuclear localization in normal cells .

What approaches can be used to develop SRRM2-targeted therapeutics?

For developing SRRM2-targeted therapies:

• CAR-T cell development:

  • Design CAR constructs with antibody variable regions in optimal orientation (VH→VL vs. VL→VH)

  • Include appropriate co-stimulatory domains (e.g., 4-1BB/CD137 and CD3-ζ)

  • Test functionality through cytokine secretion (IFN-γ, TNF-α) and cytotoxicity assays

  • SRRM2 CAR-T cells have shown significantly elevated secretion of IFN-γ and TNF-α and enhanced cytotoxicity against AML cell lines

  • In AML mouse models, SRRM2 CAR-T cells displayed significant suppression of AML and extended survival

• Antibody-based therapies:

  • Develop antibody-drug conjugates targeting surface SRRM2

  • Optimize antibody properties for therapeutic applications

  • Engineer formats suitable for clinical use (humanized or fully human antibodies)

• Validation approaches:

  • Assess potential off-target effects on normal tissues

  • Confirm cancer specificity through comprehensive testing

  • Develop companion diagnostics to identify patients with high surface SRRM2 expression

Research has demonstrated that SRRM2-specific antibodies can serve as a basis for developing new targeted cancer therapies .

What are the technical challenges in developing SRRM2 antibodies for research and therapeutic applications?

Key challenges include:

• Structural complexities:

  • SRRM2 is a large protein (~300 kDa) with multiple domains

  • Contains intrinsically disordered regions that may complicate epitope recognition

  • Different epitopes may be exposed in different cellular locations

• Specificity considerations:

  • Ensuring antibodies specifically bind surface-exposed SRRM2 on cancer cells

  • Avoiding cross-reactivity with nuclear SRRM2 in normal cells

  • Distinguishing SRRM2 from other SR proteins with potential structural similarities

• Validation requirements:

  • Need for multi-method validation approaches

  • Confirmation of binding in diverse cancer types

  • Verification of therapeutic efficacy in both in vitro and in vivo models

• Therapeutic development:

  • Optimizing antibody parameters for clinical applications

  • Developing effective CAR-T cell constructs with appropriate signaling domains

  • Balancing efficacy with safety considerations

Researchers have addressed these challenges through careful validation using techniques like immunoprecipitation followed by proteomic analysis .

What flow cytometry protocols are recommended for detecting surface SRRM2?

For flow cytometry detection of surface SRRM2:

• Cell preparation:

  • Harvest cells using gentle methods to preserve surface proteins

  • Avoid permeabilization for surface-only detection

  • If detecting both surface and intracellular SRRM2, prepare parallel samples with and without permeabilization

• Staining protocol:

  • Use validated SRRM2-specific antibodies at optimized concentrations

  • Include appropriate isotype controls

  • For multiple myeloma studies, combine with plasma cell markers for accurate identification

• Analysis parameters:

  • Quantify SRRM2 expression as percentage of positive cells

  • Measure mean fluorescence intensity to assess expression levels

  • Compare surface expression between patient samples and controls

This approach has been successfully used to correlate SRRM2 expression on MM plasma cells with clinical outcomes and risk stratification .

How can SRRM2 antibodies be used to monitor treatment response in cancer patients?

SRRM2 antibody-based monitoring shows promise:

• Expression patterns across treatment phases:

  • In newly diagnosed MM: 82.9% positivity rate (29/35 patients)

  • In VGPR+PR subgroup: 80% positivity (4/5 samples)

  • In SD+PD subgroup: 92.9% positivity (26/28 cases)

  • In PCL subgroup: 100% positivity (11/11 cases)

  • In CR subgroup: only 22.2% positivity (2/9 samples)

• Methodological approach:

  • Use flow cytometry to quantify SRRM2 expression on cancer cells

  • Track changes in expression levels during treatment

  • Correlate with clinical response and disease progression

  • Consider SRRM2 expression levels in treatment decision-making

• Clinical correlation:

  • High SRRM2 expression correlates with worse treatment outcomes

  • Can help identify high-risk patients who might benefit from more intensive therapy

  • May serve as an early indicator of treatment resistance

This evidence suggests SRRM2 detection using specific antibodies can be valuable for monitoring treatment efficacy and predicting outcomes .

What methodological considerations are important for immunohistochemical detection of SRRM2?

For immunohistochemical detection of SRRM2:

• Sample processing:

  • For nuclear SRRM2: Standard fixation and permeabilization

  • For surface SRRM2: Consider modified protocols to preserve surface epitopes

  • The large size and partially disordered structure of SRRM2 may require optimized fixation conditions

• Antibody selection:

  • Use validated antibodies with confirmed specificity

  • Consider the target epitope accessibility in processed tissues

• Protocol optimization:

  • Test different antigen retrieval methods

  • Optimize antibody concentration and incubation times

  • Include appropriate positive and negative controls

• Interpretation:

  • Evaluate both staining intensity and pattern

  • Distinguish between nuclear and membrane staining

  • Compare with normal tissue controls

Immunohistochemical staining has successfully demonstrated high SRRM2 expression in the bone marrow of MM patients with high plasma cell infiltration .

How might SRRM2 surface expression relate to cancer progression mechanisms?

While the direct mechanisms remain under investigation, several hypotheses emerge:

• Potential functional roles:

  • Surface SRRM2 might interact with the tumor microenvironment

  • Could potentially function as a signaling molecule

  • May contribute to cancer cell immune evasion

  • Could affect cell adhesion or migration properties

• Association with disease progression:

  • High SRRM2 expression correlates with worse outcomes in multiple cancers

  • Associated with high-risk cytogenetic features such as 1q21 amplification in MM

  • Correlation with treatment resistance suggests potential functional roles

• Relationship to cellular processes:

  • As an RNA splicing factor, surface expression might reflect broader dysregulation of RNA processing in cancer

  • Could relate to stress response mechanisms in cancer cells

  • May be linked to exosome/extracellular vesicle biology

This represents a promising area for future research that could provide insights into fundamental cancer biology.

What methodological approaches are recommended to study SRRM2 in patient samples?

For patient sample studies:

• Fresh sample processing:

  • Process samples promptly to preserve protein integrity

  • Use standardized protocols for consistent results across samples

  • Consider both flow cytometry and immunohistochemistry approaches

• Biobanked samples:

  • Evaluate SRRM2 stability in stored samples

  • Optimize protocols for frozen or fixed archived specimens

  • Include appropriate controls for storage-related variations

• Multi-parameter analysis:

  • Combine SRRM2 detection with other prognostic markers

  • Correlate with clinical data including treatment response

  • Analyze SRRM2 in context of molecular and cytogenetic profiles

• Data interpretation:

  • Consider SRRM2 expression in risk stratification models

  • Evaluate potential as a predictive biomarker for specific therapies

  • Assess changes in expression during disease progression

These approaches have successfully demonstrated SRRM2's value as a biomarker in MM and AML .

How do the methods for studying nuclear versus surface SRRM2 differ?

The methodological approaches differ significantly:

• Sample preparation distinctions:

  • Nuclear SRRM2: Requires permeabilization to access nuclear compartment

  • Surface SRRM2: Must avoid permeabilization to specifically detect surface expression

  • For comparative studies, parallel samples should be processed with both methods

• Technical approach variations:

  • Nuclear SRRM2: Standard nuclear protein detection methods apply

  • Surface SRRM2: Requires techniques optimized for membrane proteins

  • Different antibody concentrations may be optimal for each localization

• Controls and validation:

  • Nuclear SRRM2: Compare with other nuclear speckle proteins

  • Surface SRRM2: Include membrane protein controls

  • Use normal cells as negative controls for surface expression

• Interpretation considerations:

  • Nuclear SRRM2: Evaluate in context of splicing functions

  • Surface SRRM2: Assess in context of cancer progression and therapeutic targeting

  • Consider potential functional differences between nuclear and surface forms

These methodological distinctions are critical for accurate interpretation of SRRM2 biology in normal and cancer contexts .

What are the most promising approaches for developing SRRM2-targeted precision medicine?

Several promising approaches emerge:

• Immunotherapeutic strategies:

  • CAR-T cell therapy: SRRM2-specific CAR-T cells have shown significant efficacy in preclinical models

  • Antibody-drug conjugates: Leveraging SRRM2 antibodies to deliver cytotoxic payloads

  • Bispecific antibodies: Engaging immune cells with cancer cells expressing surface SRRM2

• Diagnostic applications:

  • Companion diagnostics to identify patients likely to benefit from SRRM2-targeted therapies

  • Prognostic biomarker for risk stratification across multiple cancer types

  • Monitoring marker for treatment response and disease progression

• Combinatorial approaches:

  • Combining SRRM2-targeted therapies with standard treatments

  • Exploring synergies with other immunotherapeutic approaches

  • Sequential treatment strategies based on SRRM2 expression patterns

Given the cancer-specific surface expression of SRRM2 across multiple cancer types, these approaches have significant potential for clinical translation .

What methodological questions remain unresolved in SRRM2 antibody research?

Key unresolved questions include:

• Epitope characterization:

  • Which SRRM2 epitopes are accessible on the cancer cell surface?

  • How do surface-exposed epitopes differ from those in nuclear SRRM2?

  • Which epitopes are optimal for therapeutic targeting?

• Antibody engineering challenges:

  • Optimizing antibody properties for therapeutic applications

  • Developing formats with improved tumor penetration

  • Balancing affinity with specificity for cancer-selective targeting

• Methodological standardization:

  • Developing standardized protocols for SRRM2 detection across laboratories

  • Establishing threshold values for clinical decision-making

  • Creating reference standards for quantitative assessments

• Functional characterization:

  • Methods to determine the functional significance of surface SRRM2

  • Techniques to explore interactions between surface SRRM2 and the tumor microenvironment

  • Approaches to study potential signaling roles of surface SRRM2

Addressing these questions will be crucial for advancing SRRM2 antibody applications in both research and clinical settings .