MORF4L2 Antibody

What cellular functions is MORF4L2 involved in and how are antibodies used to study them?

MORF4L2 is a component of the NuA4 histone acetyltransferase complex involved in transcriptional activation of select genes principally by acetylation of nucleosomal histone H4 and H2A . This modification alters nucleosome-DNA interactions and promotes interaction of modified histones with transcription-regulating proteins. Research indicates MORF4L2 is critical for the activation of transcriptional programs associated with:

• Oncogene and proto-oncogene mediated growth induction

• Tumor suppressor mediated growth arrest

• Replicative senescence

• Apoptosis mechanisms

• DNA repair processes

MORF4L2 antibodies enable researchers to detect this protein in various experimental contexts through multiple techniques including Western blotting, immunofluorescence, immunohistochemistry, and ELISA. These methods allow visualization of MORF4L2's localization (primarily nuclear), measurement of expression levels, and investigation of protein-protein interactions within the NuA4 complex .

What is the expected molecular weight of MORF4L2 in Western blot applications?

MORF4L2 has a calculated molecular weight of approximately 32 kDa, but the observed molecular weight in Western blot applications typically falls between 30-32 kDa . This slight discrepancy between calculated and observed weights may be attributed to post-translational modifications or protein folding characteristics. When conducting Western blot analysis with MORF4L2 antibodies, researchers should expect to observe bands in this range when using appropriate reducing conditions and separation techniques .

The antibody dilution recommendations for Western blot applications generally range from 1:500 to 1:1000, though this may vary between specific antibody products . Positive Western blot detection has been confirmed in various cell lines including HeLa, A431, A549, and MCF-7 cells, as well as in human and mouse liver tissues .

What species cross-reactivity should researchers expect with MORF4L2 antibodies?

Cross-reactivity of MORF4L2 antibodies varies based on the specific product and immunogen used. Most commercially available antibodies show high reactivity with human MORF4L2 samples . Many antibodies also demonstrate cross-reactivity with mouse and rat MORF4L2, making them suitable for comparative studies across these common model organisms .

Sequence homology analysis through BLAST reveals the following cross-reactivity potential by species:

• Human: 100% identity

• Mouse, Rat, Dog, Horse: approximately 92% identity

• Bovine: 84% identity

• Other species (Pig, Rabbit, Guinea Pig): variable identity ranging from 84-92%

When selecting a MORF4L2 antibody for cross-species applications, researchers should verify the specific cross-reactivity profile of their chosen antibody and consider sequence homology in the immunogen region.

How is MORF4L2 implicated in cancer progression and immune cell infiltration?

Recent research has revealed significant associations between MORF4L2 expression and cancer progression. Studies indicate that high expression of MORF4L2 is associated with worse clinical features and increased macrophage infiltration in breast cancer tissues . Specifically, MORF4L2 has been found to induce immunosuppressive microenvironments and contribute to immunotherapy resistance through the GRHL2/MORF4L2/H4K12Ac/CSF1 axis in triple-negative breast cancer .

Correlation analyses have demonstrated that MORF4L2 expression exhibits:

• Strong negative association with CD8+ T cells

• Positive correlation with M2 macrophages and resting mast cells

• Consistent negative correlation with CD8+ T cells alongside other progression-related DNA damage repair genes (GNPNAT1 and VAV3)

Immunohistochemistry sections have shown significant co-localization of positively stained regions of MORF4L2 with CD68, a marker of macrophages, while few CD8+ T cells were detected within these regions . Functional studies have revealed that knockdown of MORF4L2 resulted in impaired proliferation of MCF-7 breast cancer cells, suggesting its direct role in promoting cancer cell growth .

What is the relationship between MORF4L2 and miR-3156-5p in disease contexts?

Research has uncovered an important inverse relationship between MORF4L2 and miR-3156-5p expression in the context of multiple endocrine neoplasia type 1 (MEN1). Studies have shown that miR-3156-5p is significantly downregulated in MEN1 patients compared to matched unaffected relatives .

In vitro functional studies in BON-1 pancreatic neuroendocrine tumor cells demonstrated that:

• Transfection with miR-3156-5p mimic significantly reduced MORF4L2 protein expression by 46% (p<0.005)

• Treatment with miR-3156-5p inhibitor significantly increased MORF4L2 expression 1.5-fold (p<0.05)

• Similar regulatory effects were observed in HEPG2 cells

These findings suggest that miR-3156-5p directly regulates MORF4L2 expression, and this relationship may serve as a potential serum biomarker for neuroendocrine tumor occurrence in MEN1 patients. This inverse relationship contributes to the reliability of non-invasive biomarker approaches for monitoring tumor development and progression .

How can researchers validate MORF4L2 antibody specificity for their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research results. For MORF4L2 antibodies, a multi-faceted validation approach is recommended:

• Positive and negative control samples: Use cell lines with known MORF4L2 expression levels. HeLa, MCF-7, and A549 cells are documented to express MORF4L2 and serve as positive controls .

• Recombinant protein validation: Many suppliers provide recombinant MORF4L2 protein fragments (PrEST Antigens) that can be used as positive controls in Western blot applications .

• siRNA knockdown validation: Transfect cells with MORF4L2-specific siRNAs to create a negative control. Studies have shown successful gene silencing of MORF4L2 in MCF-7 cells, resulting in reduced expression that can be verified by antibody-based detection methods .

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of MORF4L2 to confirm that the observed signal is specific to the target protein rather than cross-reactivity with other proteins.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide prior to application in the detection method. If the signal disappears or is significantly reduced, this supports antibody specificity .

What are the optimal conditions for MORF4L2 detection in different applications?

Optimizing experimental conditions for MORF4L2 detection varies by application:

Western Blotting (WB):

• Recommended dilution: 1:500-1:1000

• Sample preparation: Complete cell lysis in RIPA buffer with protease inhibitors

• Expected molecular weight: 30-32 kDa

• Blocking: 5% non-fat milk or BSA in TBST (sample-dependent)

• Secondary antibody: Anti-rabbit IgG (for rabbit-hosted primary antibodies)

• Detection method: ECL chemiluminescence

Immunofluorescence (IF)/Immunocytochemistry (ICC):

• Recommended dilution: 1:50-1:500

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1-0.5% Triton X-100 for 10 minutes

• Blocking: 1-5% BSA or normal serum

• Positive detection confirmed in: MCF-7 cells, HeLa cells

• Nuclear counterstain: DAPI recommended due to nuclear localization of MORF4L2

Immunohistochemistry (IHC):

• Fixation: Formalin-fixed, paraffin-embedded tissues

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: Endogenous peroxidase blocking with 3% H₂O₂ followed by protein blocking

• Detection systems: HRP-polymer conjugated secondary antibodies with DAB substrate

• Counterstain: Hematoxylin for nuclear contrast

What controls should be included when designing experiments with MORF4L2 antibodies?

Robust experimental design for MORF4L2 antibody applications should include several types of controls:

Positive Controls:

• Cell lines with confirmed MORF4L2 expression (HeLa, MCF-7, A549, A431)

• Human liver tissue (shown to express MORF4L2)

• Recombinant MORF4L2 protein fragments (for Western blot applications)

Negative Controls:

• Primary antibody omission control

• Isotype control (rabbit IgG for rabbit-hosted primary antibodies)

• siRNA-treated cells with MORF4L2 knockdown

• Non-expressing tissues or cell lines (tissue-specific, must be validated)

Technical Controls:

• Loading control for Western blot (e.g., tubulin, GAPDH, or β-actin)

• Internal control for IHC (tissue with known expression pattern)

• Background fluorescence control for IF/ICC

Validation Controls:

• Multiple antibodies targeting different MORF4L2 epitopes

• Correlation of protein detection with mRNA expression data

• Peptide competition assay with immunizing peptide

How should researchers approach MORF4L2 co-localization studies with other proteins?

When investigating MORF4L2's interactions with other proteins, particularly components of the NuA4 complex or in cancer research contexts, the following methodological approaches are recommended:

Double Immunofluorescence Staining:

• Use antibodies raised in different host species (e.g., rabbit anti-MORF4L2 and mouse anti-CD68)

• Apply primary antibodies sequentially or simultaneously (protocol-dependent)

• Use spectrally distinct fluorophore-conjugated secondary antibodies

• Include single-stained controls to assess bleed-through

• Quantify co-localization using correlation coefficients (e.g., Pearson's, Manders')

Proximity Ligation Assay (PLA):

• More sensitive for detecting protein-protein interactions

• Provides higher spatial resolution than conventional co-localization

• Generates punctate signals only when proteins are within 40nm proximity

• Useful for confirming direct interactions between MORF4L2 and other NuA4 complex components

Co-Immunoprecipitation (Co-IP):

• Use MORF4L2 antibodies for pull-down experiments

• Western blot for interacting proteins or mass spectrometry for unbiased detection

• Include IgG control immunoprecipitation

• Consider crosslinking for transient interactions

Research has shown successful co-localization studies between MORF4L2 and immune cell markers such as CD68 (macrophage marker), demonstrating the utility of these techniques for investigating MORF4L2's role in modulating the tumor microenvironment .

What are common challenges when using MORF4L2 antibodies and how can they be addressed?

Challenge: Non-specific bands in Western blot

• Solution: Optimize antibody dilution (start with 1:500-1:1000)

• Increase blocking time/concentration (5% milk or BSA for 2 hours)

• Try different blocking agents (switch between milk and BSA)

• Increase washing frequency and duration

• Use gradient gels for better separation around 30-32 kDa region

Challenge: Weak signal in immunohistochemistry

• Solution: Optimize antigen retrieval methods (try both citrate and EDTA buffers)

• Extend primary antibody incubation (overnight at 4°C)

• Use signal amplification systems (biotin-streptavidin or tyramide)

• Test different fixation protocols for better epitope preservation

• Decrease antibody dilution (try 1:50-1:100 range)

Challenge: High background in immunofluorescence

• Solution: Increase blocking time/concentration

• Add 0.1-0.3% Triton X-100 to antibody diluent

• Pre-absorb primary antibody with tissue powder

• Use more stringent washing (0.1% Tween-20 in PBS)

• Try fluorophore-conjugated Fab fragments instead of whole IgG secondaries

Challenge: Inconsistent results across tissue types

• Solution: Optimize fixation time for each tissue type

• Adjust antigen retrieval time based on tissue density

• Use tissue-specific blocking agents to reduce non-specific binding

• Consider using automated staining platforms for consistency

• Validate antibody performance in each tissue type independently

How should researchers interpret discrepancies between MORF4L2 protein and mRNA expression?

Discrepancies between MORF4L2 protein and mRNA expression are not uncommon and may reflect important biological processes. Consider these interpretive approaches:

• Post-transcriptional regulation: Research has demonstrated that miR-3156-5p directly regulates MORF4L2 protein expression without necessarily affecting mRNA levels . When interpreting discrepancies, consider:

  • Analyzing miRNA expression profiles, particularly miR-3156-5p

  • Examining mRNA stability through actinomycin D chase experiments

  • Investigating RNA-binding proteins that might regulate MORF4L2 mRNA

• Protein stability differences: MORF4L2 protein stability may vary across:

  • Different cell types or tissue contexts

  • Normal versus pathological states

  • Response to treatment conditions

  • Investigate using proteasome inhibitors (MG132) to assess degradation rates

• Technical considerations:

  • Verify antibody specificity using multiple validation approaches

  • Compare results from different antibodies targeting distinct epitopes

  • Ensure adequate controls for both protein and mRNA detection methods

  • Consider absolute quantification methods for both protein and mRNA

• Biological relevance:

  • In some cancer contexts, MORF4L2 protein may be elevated while mRNA remains unchanged

  • This could indicate post-transcriptional regulatory mechanisms are important in disease progression

  • Focus on protein level changes for functional studies and clinical correlations

How can MORF4L2 expression data be integrated with immune cell infiltration analyses in cancer research?

Recent studies have revealed significant correlations between MORF4L2 expression and immune cell infiltration patterns in cancer tissues . To effectively integrate these datasets:

• Sequential or multiplex staining approaches:

  • Perform sequential IHC for MORF4L2 and immune cell markers (CD8, CD68)

  • Use multiplex immunofluorescence to simultaneously visualize MORF4L2 and multiple immune markers

  • Quantify co-localization or spatial relationships between MORF4L2+ cells and immune cells

• Computational analysis integration:

  • Apply CIBERSORT algorithm to deconvolute immune cell populations from bulk RNA-seq data

  • Correlate MORF4L2 expression levels with specific immune cell abundances

  • Studies have shown MORF4L2 has strong negative correlation with CD8+ T cells and positive correlation with M2 macrophages

• Functional validation experiments:

  • Design co-culture experiments with MORF4L2-modulated cancer cells and immune cells

  • Measure immune cell recruitment, activation, and function

  • Assess cytokine/chemokine profiles in relation to MORF4L2 expression

  • Investigate the GRHL2/MORF4L2/H4K12Ac/CSF1 axis implicated in immunotherapy resistance

• Single-cell analysis approaches:

  • Perform single-cell RNA-seq to correlate MORF4L2 expression with immune cell states

  • Use CyTOF or single-cell proteomics to examine protein-level correlations

  • Integrate spatial transcriptomics to map MORF4L2 expression in the tumor microenvironment

Recent findings demonstrate that MORF4L2 may induce immunosuppressive microenvironments through specific molecular pathways, particularly in triple-negative breast cancer . Understanding these relationships can help develop more effective immunotherapy strategies and identify patients who might benefit from targeted approaches.

How might MORF4L2 serve as a biomarker in cancer and other diseases?

MORF4L2 shows promise as a biomarker in several disease contexts:

Cancer Biomarker Applications:

• High MORF4L2 expression correlates with worse clinical features in breast cancer

• Part of a DNA damage-repair-related signature that reflects microenvironmental features and therapeutic response

• May predict response to immunotherapy through its role in modulating immune cell infiltration

• Knockdown of MORF4L2 impairs proliferation of cancer cells, suggesting therapeutic targeting potential

Neuroendocrine Tumor (NET) Detection:

• The inverse relationship between miR-3156-5p and MORF4L2 represents a potential serum biomarker for NET occurrence in MEN1 patients

• This relationship could facilitate the detection of NET development through non-invasive blood testing

• The combination of these markers may improve sensitivity and specificity compared to single markers

Methodological Approaches for Biomarker Development:

• Validate expression in larger patient cohorts using tissue microarrays

• Develop serum-based detection methods for circulating MORF4L2

• Integrate MORF4L2 with other markers for improved diagnostic accuracy

• Correlate expression with treatment response and clinical outcomes

• Establish standardized detection protocols for clinical implementation

What techniques are advancing our understanding of MORF4L2's role in histone modification?

As a component of the NuA4 histone acetyltransferase complex, MORF4L2's role in histone modification is being investigated using advanced techniques:

Chromatin Immunoprecipitation (ChIP) Approaches:

• ChIP-seq to map genome-wide binding sites of MORF4L2

• CUT&RUN for higher resolution mapping with lower background

• HiChIP to connect MORF4L2 binding with 3D chromatin organization

• Re-ChIP to identify genomic regions bound by multiple NuA4 components

Histone Modification Analysis:

• ChIP-seq for H4K12Ac and other relevant histone marks in MORF4L2-modulated systems

• Mass spectrometry to characterize histone modification patterns

• Antibody-based detection of specific modifications (Western blot, IF)

• ATAC-seq to assess chromatin accessibility changes related to MORF4L2 function

Functional Genomics Approaches:

• CRISPR-Cas9 screening to identify genetic dependencies on MORF4L2

• CRISPRi/CRISPRa for precise modulation of MORF4L2 expression

• RNA-seq to characterize transcriptional programs regulated by MORF4L2

• proteomics to identify MORF4L2 interaction partners and complex components

Structural Biology Methods:

• Cryo-EM to resolve structure of MORF4L2 within the NuA4 complex

• Hydrogen-deuterium exchange mass spectrometry for protein dynamics

• Cross-linking mass spectrometry to map protein-protein interactions

Recent research has identified the GRHL2/MORF4L2/H4K12Ac/CSF1 axis as a mechanism through which MORF4L2 induces immunosuppressive microenvironments in triple-negative breast cancer , highlighting the importance of understanding MORF4L2's role in histone modification for therapeutic development.