MORF4L1 Antibody

What is MORF4L1 and what is its biological significance?

MORF4L1 (also known as MRG15) is a member of a subgroup of histone acetyltransferases and belongs to the mortality factor on chromosome 4 (MORF4) class of proteins. It shares 96% homology with MORF4 in amino acid sequences . Unlike most histone acetyltransferases that activate gene transcription and promote cell proliferation, MORF4L1 has been shown to function as a tumor suppressor in several cancers .

MORF4L1 plays critical roles in:

• Chromatin remodeling and transcriptional regulation during embryonic development

• Mediating epithelial cell death in certain conditions

• DNA repair through homology-directed repair of chromosomal breaks

• Tumor suppression by increasing p21 and E-cadherin levels

Studies have shown that MORF4L1 expression is frequently decreased in several cancers, including nasopharyngeal carcinoma (NPC), with methylation of its promoter significantly higher in tumor cells compared to normal cells .

What are the primary applications for MORF4L1 antibodies in research?

MORF4L1 antibodies serve multiple crucial applications in molecular and cellular research:

• Western Blotting (WB): The most common application, allowing detection of MORF4L1 protein expression levels in cell and tissue lysates. Commercial antibodies typically recommend dilutions between 1:500-1:2000 for WB applications .

• Immunoprecipitation (IP): Used to isolate MORF4L1 and its interacting partners from complex protein mixtures, with typical dilutions of 1:50 .

• ELISA: For quantitative measurement of MORF4L1 protein levels .

These applications enable researchers to investigate MORF4L1's expression patterns, protein-protein interactions, and its role in various cellular processes and disease states.

What are the key specifications to consider when selecting a MORF4L1 antibody?

When selecting a MORF4L1 antibody for research, several critical specifications should be considered:

Some antibodies may recognize a 90 kDa protein of unknown origin in certain cells, which should be considered when interpreting results . Additionally, checking if the antibody recognizes specific MORF4L1 isoforms is important, as at least 5 isoforms have been identified .

How can I validate the specificity of my MORF4L1 antibody?

Validating antibody specificity is crucial for generating reliable research data. For MORF4L1 antibodies, consider these approaches:

• Positive and negative control samples: Use cell lines known to express MORF4L1 at different levels. Based on published data, MCF-7, HepG2, and THP-1 cell lines serve as good positive controls . For tissue samples, mouse brain, spleen, and testis have been documented as positive controls .

• Knockdown/knockout validation: Use siRNA or CRISPR-Cas9 to reduce or eliminate MORF4L1 expression. The antibody signal should decrease correspondingly in knockdown samples.

• Overexpression validation: Compare wild-type cells with those overexpressing MORF4L1. A specific antibody will show increased signal intensity in overexpressing cells.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application. A specific antibody's signal should be blocked or significantly reduced.

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of MORF4L1 to confirm consistent results.

What are the optimal Western blot conditions for MORF4L1 detection?

Optimizing Western blot protocols for MORF4L1 detection requires attention to several parameters:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation status

  • Maintain samples at 4°C during preparation to prevent degradation

• Gel electrophoresis:

  • 10-12% SDS-PAGE gels are suitable for resolving the 40-41 kDa MORF4L1 protein

  • Load 20-50 μg of total protein per lane

• Transfer conditions:

  • Semi-dry or wet transfer systems both work effectively

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST

  • Primary antibody dilution: 1:1000 for commercial antibodies like Cell Signaling's D2Y4J Rabbit mAb

  • Incubate primary antibody overnight at 4°C

  • Secondary antibody: Anti-Rabbit IgG conjugated to HRP at 1:10,000 dilution for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) systems like BeyoECL Moon detection system

  • For densitometry analysis, software like ImageJ can be used

How should I troubleshoot if I'm unable to detect MORF4L1 in my experiments?

When facing challenges in MORF4L1 detection, consider these troubleshooting strategies:

• No signal in Western blot:

  • Verify sample integrity using housekeeping proteins (GAPDH, β-actin)

  • Check for proper transfer using Ponceau S staining

  • Increase protein loading (50-100 μg)

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

  • Use more sensitive detection reagents

• Multiple bands or unexpected band size:

  • MORF4L1 has multiple isoforms; confirm which isoform your antibody targets

  • Some antibodies may detect a 90 kDa protein of unknown origin

  • Post-translational modifications can alter migration patterns

  • Optimize SDS-PAGE conditions (percentage, running time)

• High background:

  • Increase washing steps (5-6 times, 5 minutes each)

  • Optimize blocking conditions (try BSA instead of milk)

  • Reduce antibody concentration

  • Use fresher reagents

• Inconsistent results across experiments:

  • Standardize sample preparation protocols

  • Prepare fresh buffers and reagents

  • Consider MORF4L1 expression variability based on cell density and culture conditions

How is MORF4L1 expression altered in cancer, and what methods are best to study these changes?

MORF4L1 expression is significantly downregulated in multiple cancer types compared to normal tissues. Based on data from MethHC (an integrated database of DNA methylation and gene expression from The Cancer Genome Atlas), MORF4L1 shows decreased expression in breast, colon, lung cancer, and nasopharyngeal carcinoma (NPC) .

Recommended methods for studying MORF4L1 expression in cancer:

• RT-qPCR: For quantifying mRNA expression levels, using primers:

  • MORF4L1 forward: 5′-AGCAATGTTGGCTTATACACCTC-3′

  • MORF4L1 reverse: 5′-AGCTTTCCGATGGTACTCAGG-3′

• Western blotting: For protein expression analysis using validated antibodies at 1:1000 dilution .

• Immunohistochemistry: For spatial distribution in tissue sections.

• Methylation analysis: Since promoter methylation appears to regulate MORF4L1 expression in cancer, techniques like bisulfite sequencing or methylation-specific PCR can provide insights into epigenetic regulation.

• Public database mining: Utilize cancer genomics databases like TCGA, CCLE, or cBioPortal to analyze MORF4L1 expression across large cohorts of cancer samples.

What experimental approaches can be used to study MORF4L1's tumor suppressor function?

To investigate MORF4L1's tumor suppressor role, researchers can employ these experimental approaches:

• Gain/loss-of-function studies:

  • Overexpression of MORF4L1 in cancer cell lines followed by functional assays

  • Knockdown using siRNA or CRISPR-Cas9 technology

• Functional assays:

  • Cell proliferation assays (MTT, BrdU incorporation)

  • Colony formation assays

  • Migration assays (wound healing, transwell)

  • Invasion assays (Matrigel-coated transwell)

  • Cell cycle analysis by flow cytometry

• Downstream target analysis:

  • Monitor p21 and E-cadherin levels, as MORF4L1 has been shown to increase their expression

  • Use RT-qPCR with primers:

    • E-cadherin F: 5′-AATAGTGCCTAAAGTGCTGC-3′

    • E-cadherin R: 5′-AGACCCACCTCAATCATCCT-3′

    • p21 F: 5′-CGATGGAACTTCGACTTTGTCA-3′

    • p21 R: 5′-GCACAAGGGTACAAGACAGTG-3′

• In vivo models:

  • Xenograft models with MORF4L1-overexpressing or knockout cancer cells

  • Patient-derived xenografts (PDXs)

• 3D culture systems:

  • Organoid cultures to better recapitulate tumor microenvironment

How can researchers investigate MORF4L1's role in chromatin remodeling and transcriptional regulation?

MORF4L1's functions in chromatin remodeling and transcriptional regulation can be investigated through several approaches:

• Chromatin immunoprecipitation (ChIP):

  • Use MORF4L1 antibodies for ChIP followed by sequencing (ChIP-seq) to identify genomic binding sites

  • ChIP-qPCR to verify binding at specific loci

• Co-immunoprecipitation (Co-IP):

  • Identify proteins that interact with MORF4L1 in chromatin remodeling complexes

  • Use antibodies at 1:50 dilution as recommended for IP applications

• Histone modification analysis:

  • Western blotting for specific histone marks in MORF4L1-depleted or overexpressing cells

  • ChIP-seq for histone modifications to assess global changes

• Transcriptome analysis:

  • RNA-seq to identify genes regulated by MORF4L1

  • RT-qPCR validation of key target genes

• Chromatin accessibility assays:

  • ATAC-seq to measure changes in chromatin accessibility upon MORF4L1 modulation

  • DNase-seq or MNase-seq as alternative approaches

• Live-cell imaging:

  • Fluorescently tagged MORF4L1 to monitor dynamic interactions with chromatin

What is known about MORF4L1's involvement in DNA repair pathways?

MORF4L1 plays a significant role in DNA repair, particularly in homology-directed repair (HDR) of chromosomal breaks:

• Known interactions: MORF4L1 interacts with key DNA repair proteins including:

  • BRCA2 (Breast Cancer Type 2 Susceptibility Protein)

  • PALB2 (Partner and Localizer of BRCA2)

  • RAD51 recombinase

  • Replication Protein A1 (RPA1)

• Functional significance: These interactions suggest MORF4L1 helps recruit or stabilize repair factors at DNA damage sites.

• Experimental approaches to study this function:

  • Immunofluorescence to detect MORF4L1 localization to DNA damage foci

  • Co-IP followed by Western blot to confirm interactions with repair proteins

  • HR reporter assays to measure HDR efficiency in MORF4L1-depleted cells

  • Laser microirradiation to track MORF4L1 recruitment to damaged DNA in real-time

• Clinical relevance: Understanding MORF4L1's role in DNA repair may provide insights into cancer susceptibility and response to DNA-damaging therapies.

How does MORF4L1 methylation status affect its function, and what techniques can measure this?

MORF4L1 promoter methylation appears to be an important regulatory mechanism, particularly in cancer contexts where higher methylation rates correlate with decreased expression . Researchers can investigate this relationship using these approaches:

• Bisulfite sequencing:

  • Gold standard for mapping methylation at single-nucleotide resolution

  • Can be performed as targeted sequencing for the MORF4L1 promoter region

• Methylation-specific PCR (MSP):

  • Faster alternative to bisulfite sequencing

  • Requires primers specific for methylated and unmethylated versions of the sequence

• Pyrosequencing:

  • Quantitative assessment of methylation at specific CpG sites

• Methylation arrays:

  • For genome-wide methylation profiling including MORF4L1

  • Examples include Illumina MethylationEPIC BeadChip

• Functional validation:

  • Treatment with DNA methyltransferase inhibitors (e.g., 5-aza-2'-deoxycytidine) to determine if demethylation restores MORF4L1 expression

  • Reporter assays with methylated and unmethylated MORF4L1 promoter constructs

What approaches can be used to study MORF4L1's post-translational modifications?

Post-translational modifications (PTMs) of MORF4L1 can significantly impact its function. Based on information from ActiveDriverDB, MORF4L1 contains 27 PTM sites . Researchers can employ these techniques to investigate MORF4L1 PTMs:

• Mass spectrometry (MS):

  • Immunoprecipitate MORF4L1 and analyze by LC-MS/MS

  • Phosphoproteomics to identify phosphorylation sites

  • SILAC-MS for quantitative comparison of PTMs under different conditions

• Phospho-specific antibodies:

  • When available, use phospho-specific antibodies for Western blotting

• Phos-tag SDS-PAGE:

  • For separation and detection of phosphorylated MORF4L1 isoforms

• Mutagenesis studies:

  • Generate point mutations at potential PTM sites (identified in databases like ActiveDriverDB )

  • Compare function of wild-type vs. mutant MORF4L1

• In vitro kinase assays:

  • Identify kinases responsible for MORF4L1 phosphorylation

• PTM-specific functional assays:

  • Compare chromatin binding, protein interactions, or transcriptional effects of modified vs. unmodified MORF4L1

This methodological framework provides researchers with comprehensive tools to explore the complex PTM landscape of MORF4L1 and its functional consequences.