MCF2L Antibody

What is MCF2L and why is it important in scientific research?

MCF2L is a guanine nucleotide exchange factor that interacts specifically with GTP-bound Rac1 and plays a significant role in the Rho/Rac signaling pathways . It catalyzes guanine nucleotide exchange on RHOA and CDC42, contributing to the regulation of these signaling pathways. MCF2L has been associated with several diseases, including osteoarthritis and various cancers, making it an important target for research . The protein becomes activated and highly tumorigenic when the N-terminus is truncated, suggesting its potential role in cancer development .

What are the primary applications of MCF2L antibodies in research?

MCF2L antibodies are valuable tools in several research applications:

• Western blotting for protein expression analysis (as demonstrated in breast cancer studies)

• Immunoprecipitation assays to study protein-protein interactions

• Chromatin immunoprecipitation (ChIP) assays to investigate DNA-protein interactions (similar to methods used with YAP in MCF2L-AS1 studies)

• Immunohistochemistry to examine tissue expression patterns

• Flow cytometry for cellular analysis

These applications help researchers investigate MCF2L's role in signaling pathways, disease processes, and cellular functions.

What should researchers consider when selecting an MCF2L antibody?

When selecting an MCF2L antibody, consider:

• Antibody specificity for human MCF2L (HGNC: 14576, UniProtKB/Swiss-Prot: O15068)

• Recognition of specific isoforms (MCF2L has multiple transcript variants from alternative splicing)

• Applications validated by the manufacturer (WB, IP, IHC, etc.)

• Species reactivity (especially important for animal models)

• Mono- vs polyclonal (each offering different advantages)

• Epitope location (N-terminal, C-terminal, or internal domains)

For detecting specific isoforms, verify which splice variants the antibody recognizes, as different isoforms may have distinct functions (e.g., isoform 5 activates CDC42, while isoform 3 does not) .

What is the optimal protocol for using MCF2L antibody in Western blotting?

For optimal Western blotting with MCF2L antibody:

• Extract proteins using SDS lysis buffer (2M thiourea, 2% DTT, 7M urea) supplemented with 1% protease inhibitors

• Separate proteins via 10% SDS-PAGE

• Transfer onto PVDF membranes

• Block with 5% nonfat milk for 2 hours at room temperature

• Incubate with primary MCF2L antibody (1:1000 dilution) overnight at 4°C

• Wash three times with TBST

• Incubate with HRP-conjugated secondary antibody (1:2000) for 2 hours at room temperature

• Develop using ECL detection system

For validation, include positive control samples with known MCF2L expression and negative controls where MCF2L is knocked down using siRNA, similar to the approach used in the MCF2L-AS1 studies .

How can MCF2L antibody be utilized in ChIP assays to study gene regulation?

For ChIP assays with MCF2L antibody:

• Cross-link protein-DNA complexes in target cells using 1% formaldehyde

• Lyse cells and sonicate chromatin to 200-500bp fragments

• Immunoprecipitate with 5μg of MCF2L antibody (similar to the protocol used for YAP ChIP)

• Include IgG control for non-specific binding assessment

• Reverse cross-links and purify DNA

• Analyze by qPCR with primers designed for suspected MCF2L binding regions

This method can help identify genes directly regulated by MCF2L or its associated complexes, providing insights into its role in transcriptional regulation networks.

What controls should be included when validating MCF2L antibody specificity?

To validate MCF2L antibody specificity:

• Positive controls: Lysates from cells known to express MCF2L (e.g., liver cells, as MCF2L shows expression in liver tissue)

• Negative controls:

  • Lysates from cells with MCF2L knockdown via siRNA (similar to the MCF2L-AS1 knockdown approach)

  • Peptide competition assay where antibody is pre-incubated with immunizing peptide

• Isoform controls: Lysates from cells expressing specific MCF2L isoforms to verify detection patterns

• Cross-reactivity controls: Assessment against the paralog MCF2L2

These controls ensure experimental results reflect actual MCF2L biology rather than non-specific interactions.

How is MCF2L implicated in hepatocellular carcinoma, and how can antibodies help investigate this?

MCF2L has been identified as playing a significant role in hepatocellular carcinoma (HCC):

• MCF2L is upregulated in HCC tissues, and its downregulation enhances HCC cell death induced by sorafenib

• Downregulation of MCF2L promotes ferroptosis (iron-dependent cell death) in HCC cells through the PI3K/mTOR pathway in a RhoA/Rac1 dependent manner

• MCF2L may be involved in sorafenib resistance mechanisms in HCC

MCF2L antibodies can help investigate these phenomena through:

• Protein expression analysis in patient samples

• Tracking changes in MCF2L levels during sorafenib treatment

• Monitoring RhoA/Rac1 pathway activation in response to MCF2L modulation

• Detecting MCF2L in ferroptosis-related protein complexes

What is the connection between MCF2L and osteoarthritis, and how can antibodies be used to study this relationship?

A variant in MCF2L (rs11842874) has been robustly associated with osteoarthritis with an odds ratio of 1.17 (95% CI: 1.11–1.23) across 19,041 OA cases and 24,504 controls of European descent . MCF2L regulates nerve growth factor (NGF), and treatment with humanized monoclonal antibodies against NGF is associated with reduced pain and improved function in knee OA patients .

Researchers can use MCF2L antibodies to:

• Study MCF2L expression in articular chondrocytes (rat models have shown expression in these cells)

• Examine MCF2L-NGF pathway interactions in OA tissues

• Investigate how the rs11842874 variant affects MCF2L protein expression or function

• Analyze MCF2L's role in cartilage homeostasis and degradation

How do MCF2L-AS1 and MCF2L interact in cancer development, and how can this be studied using antibodies?

MCF2L-AS1 is a long non-coding RNA that has been implicated in both hepatocellular carcinoma and breast cancer , but its relationship with MCF2L protein requires further investigation:

• In HCC, MCF2L-AS1 is upregulated and promotes cell proliferation, migration, and invasion while reducing apoptosis

• In breast cancer, high MCF2L-AS1 expression is associated with poor patient outcomes

• The functional relationship between MCF2L-AS1 and MCF2L protein remains unclear

To study their potential interaction:

• Use MCF2L antibody in conjunction with MCF2L-AS1 knockdown/overexpression to assess effects on protein levels

• Perform RNA immunoprecipitation with MCF2L antibody to test direct binding to MCF2L-AS1

• Co-localization studies using MCF2L antibody and MCF2L-AS1 RNA FISH to determine spatial relationships

• Compare expression patterns in clinical samples using MCF2L antibody (for protein) and qRT-PCR (for MCF2L-AS1)

How can MCF2L antibody be used to study the RhoA/Rac1 signaling pathway?

MCF2L functions as a guanine nucleotide exchange factor for RHOA and CDC42 , playing a crucial role in RhoA/Rac1 signaling. Researchers can use MCF2L antibodies to:

• Perform co-immunoprecipitation experiments to identify MCF2L binding partners within the signaling cascade

• Develop proximity ligation assays to visualize interactions between MCF2L and RhoA/Rac1 in situ

• Conduct activity assays after MCF2L immunoprecipitation to assess GEF activity on RhoA and CDC42

• Analyze MCF2L localization during RhoA/Rac1 pathway activation using immunofluorescence

• Study how MCF2L downregulation affects ferroptosis through RhoA/Rac1-dependent PI3K/mTOR pathway modulation

These approaches can help elucidate how MCF2L contributes to cellular processes through these signaling pathways.

How can researchers differentiate between MCF2L isoforms using antibodies?

MCF2L undergoes alternative splicing to produce multiple transcript variants . To differentiate between isoforms:

• Select antibodies raised against isoform-specific regions (particularly important as isoforms have different functions - isoform 5 activates CDC42 while isoform 3 does not)

• Perform Western blotting with positive controls for each isoform to establish expected band patterns

• Use immunoprecipitation followed by mass spectrometry to identify specific isoforms

• Combine with siRNA knockdown targeting specific exons to confirm antibody specificity

• Design validation experiments based on known functional differences (e.g., CDC42 activation assays)

Understanding isoform-specific functions is critical as they may have distinct roles in normal physiology and disease.

What techniques can be used to study MCF2L phosphorylation and post-translational modifications?

To study MCF2L post-translational modifications:

• Immunoprecipitate MCF2L using specific antibodies and analyze by:

  • Phospho-specific antibodies in Western blots

  • Mass spectrometry to identify modification sites

  • Phos-tag gel electrophoresis to separate phosphorylated forms

• Use phosphatase treatment of immunoprecipitated MCF2L to confirm phosphorylation status

• Perform kinase assays with immunopurified MCF2L to identify regulatory kinases

• Develop site-specific phospho-antibodies for key regulatory sites

• Combine with functional assays (GEF activity, protein interactions) to determine the impact of modifications

These studies can reveal regulatory mechanisms controlling MCF2L activity in signaling pathways and disease processes.

What are common causes of inconsistent results when using MCF2L antibody, and how can they be addressed?

Common causes of inconsistency include:

• Antibody degradation - Store according to manufacturer recommendations; aliquot to avoid freeze-thaw cycles

• Sample preparation issues - Use standardized lysis buffers with protease inhibitors as described in protocols

• Variable MCF2L expression levels - Include positive controls from cells known to express MCF2L

• Isoform detection differences - Confirm which isoforms your antibody detects

• Post-translational modifications - Consider phosphatase treatment if modifications affect epitope recognition

• Cross-reactivity with MCF2L2 (paralog) - Validate specificity using knockout/knockdown controls

Thorough validation using the controls described in section 2.3 can help identify and address these issues.

How can researchers address non-specific binding when using MCF2L antibody?

To minimize non-specific binding:

• Optimize blocking conditions (5% nonfat milk or BSA for 2 hours has been effective in similar studies)

• Titrate antibody concentration to find optimal dilution (starting with 1:1000 for Western blotting)

• Include additional washing steps with varying stringency

• Pre-adsorb antibody with cell/tissue lysates from MCF2L-negative samples

• For immunohistochemistry, include antigen retrieval optimization

• Perform peptide competition assays to confirm specificity

• Include appropriate negative controls (IgG and MCF2L knockdown samples)

Each application may require specific optimization to reduce background while maintaining specific signal.

How should researchers interpret conflicting data from different MCF2L antibodies?

When facing conflicting results:

• Compare antibody epitopes - different antibodies may recognize different domains or isoforms

• Verify results using orthogonal methods (e.g., mass spectrometry, mRNA expression)

• Test antibodies on known positive and negative controls

• Consider the context - tissue/cell type may affect MCF2L expression patterns

• Evaluate the potential impact of post-translational modifications on epitope accessibility

• Assess experimental conditions that might affect results (fixation methods, buffer composition)

• Consult literature for validated antibodies in your specific application

A multi-antibody approach targeting different epitopes can provide more reliable results when studying complex proteins like MCF2L.