mysm1 Antibody

Introduction to MYSM1 Antibody

MYSM1 antibodies are immunological reagents specifically designed to detect and analyze the MYSM1 protein (Myb-like, SWIRM, and MPN domains-containing protein 1), a metalloprotease with deubiquitinase activity that plays crucial roles in regulating hematopoietic stem cell function, blood cell production, and immune response. These antibodies have emerged as vital tools for investigating MYSM1's diverse biological functions across multiple physiological systems and pathological conditions .

The development of specific and sensitive MYSM1 antibodies has enabled researchers to characterize the protein's expression patterns, subcellular localization, and functional interactions, thereby advancing our understanding of MYSM1's roles in normal development and disease. These antibodies are commercially available from several manufacturers with various specifications tailored to different experimental applications.

Structure and Properties of MYSM1 Protein

Before delving into MYSM1 antibodies specifically, it is essential to understand the structure and properties of their target protein. MYSM1 belongs to the peptidase M67A family and MYSM1 subfamily . It is a metalloprotease with deubiquitinase catalytic activity, featuring characteristic SANT and SWIRM domains that facilitate protein-protein and protein-DNA interactions .

MYSM1 primarily functions as a histone H2A deubiquitinase, removing monoubiquitin from histone H2A, which is a specific epigenetic tag associated with transcriptional repression. This deubiquitination activity leads to the phosphorylation and dissociation of histone H1 from the nucleosome, thereby promoting gene activation . MYSM1 localizes predominantly to the nucleus, where it regulates gene expression through both its enzymatic activity and non-catalytic interactions with transcriptional regulators .

Recent research has also identified a cytosolic form of MYSM1 that regulates innate immunity signaling pathways by deubiquitinating components such as TRAF3, TRAF6, and RIP2 . This dual localization reflects MYSM1's multifaceted roles in cellular processes.

Validation Methods and Quality Control

Rigorous validation is essential to ensure the specificity and reliability of MYSM1 antibodies. Manufacturers employ various validation methods, including:

Western Blot Validation

Western blot validation is a primary method for demonstrating antibody specificity. For MYSM1 antibodies, this typically shows detection of a protein band at approximately 95-110 kDa, corresponding to the predicted molecular weight of MYSM1 .

Advanced validation includes comparative Western blot analysis using MYSM1 knockout cell lines. For example, Abcam's anti-MYSM1 antibody [EPR18657] was validated using wild-type and MYSM1 knockout HEK-293T cell lysates, confirming specificity by the absence of signal in the knockout samples .

Immunoprecipitation

Immunoprecipitation assays demonstrate the antibody's ability to bind native MYSM1 protein in cell lysates. For instance, MYSM1 was successfully immunoprecipitated from HeLa cell lysate using Abcam's antibody, with subsequent Western blot confirming the identity of the precipitated protein .

Cell and Tissue Staining

Immunohistochemistry and immunofluorescence validate the antibody's ability to detect MYSM1 in fixed tissues and cells, providing information about its subcellular localization. These techniques have revealed MYSM1's predominant nuclear localization in various cell types, consistent with its role in transcriptional regulation .

Technical Applications of MYSM1 Antibodies

MYSM1 antibodies have been employed in numerous technical applications across various research fields:

Western Blotting

Western blot protocols for MYSM1 antibodies typically involve:

• Sample preparation from whole cell lysates or tissue homogenates

• Protein separation on 12% SDS-PAGE gels

• Transfer to nitrocellulose membranes

• Blocking with 3-5% milk or BSA in TBS-T

• Primary antibody incubation (typically 1:500-1:2000 dilution) overnight at 4°C

• Secondary antibody incubation with appropriate HRP or fluorophore conjugates

• Development using enhanced chemiluminescence or fluorescence imaging

Immunoprecipitation

For immunoprecipitation applications, MYSM1 antibodies are typically used at concentrations of 1:100 dilution with protein A/G agarose beads to pull down endogenous or tagged MYSM1 protein from cell lysates. This technique has been valuable for studying MYSM1's protein-protein interactions .

Immunohistochemistry and Immunofluorescence

These techniques have been crucial for analyzing MYSM1 expression patterns across different tissues and cellular compartments. Studies have used MYSM1 antibodies for immunofluorescence staining to demonstrate that MYSM1 is predominantly increased in cardiomyocytes (α-actinin+ cells) rather than in fibroblasts or macrophages during cardiac injury .

Research Applications of MYSM1 Antibodies

MYSM1 antibodies have facilitated groundbreaking research across multiple biological systems:

Hematopoietic System Research

MYSM1 antibodies have been instrumental in elucidating MYSM1's role in hematopoietic stem cell (HSC) function and development. Research using these antibodies has demonstrated that MYSM1 is essential for maintaining HSC quiescence and survival .

A key study revealed that MYSM1 conditional knockout mice showed decreased bone marrow cellularity and abnormalities in HSCs and their progenitors. Immunological techniques using MYSM1 antibodies showed that MYSM1 deficiency drives HSCs from quiescence into rapid cycling, resulting in impaired engraftment and elevated reactive oxygen species (ROS) levels .

B-cell Development and Antibody Production

Research employing MYSM1 antibodies has revealed MYSM1's critical role in B-cell development and antibody production. Interestingly, studies showed that MYSM1-deficient mice, despite having severe defects in B-cell development, exhibit enhanced antibody responses against both T-cell-dependent and independent antigens .

Mechanistic investigations demonstrated that MYSM1 intrinsically represses plasma cell differentiation and antibody production by activating Pax5, a repressor of plasma cell differentiation. The following table summarizes key findings from these studies:

These findings highlight MYSM1's dual role in promoting B-cell development while repressing plasma cell differentiation and antibody production, suggesting potential therapeutic implications for modulating antibody responses .

Natural Killer Cell Development

MYSM1 antibodies have contributed to understanding MYSM1's role in Natural Killer (NK) cell development. Research has identified that MYSM1 is required for NK cell maturation but not for NK lineage specification and commitment .

Mechanistic studies using MYSM1 antibodies revealed that MYSM1 interacts with nuclear factor IL-3 (NFIL3, also known as E4BP4) to regulate the expression of inhibitor of DNA-binding protein (ID2), a critical factor for NK cell development. MYSM1 antibodies were used in co-immunoprecipitation experiments to demonstrate this protein-protein interaction .

Cancer Research

MYSM1 antibodies have been employed in cancer research, revealing diverse roles of MYSM1 across different cancer types:

Triple-Negative Breast Cancer (TNBC)

Studies using MYSM1 antibodies demonstrated that MYSM1 expression was drastically decreased in breast cancer, especially in TNBC. Functional analyses showed that MYSM1 overexpression increased cisplatin-induced apoptosis, potentially through RSK3 inactivation and decreased phosphorylation of BAD (Ser 112) .

The following findings were reported in TNBC cells with manipulated MYSM1 expression:

These findings suggest MYSM1's potential role as a tumor suppressor in TNBC .

Colorectal Cancer (CRC)

MYSM1 antibodies used in immunohistochemistry of tissue microarrays revealed that MYSM1 expression was significantly higher in adjacent normal tissues and lower in lymph node metastatic carcinomas than in primary adenocarcinomas. The research demonstrated that MYSM1 was negatively associated with CRC stage and tumor stage, suggesting its potential prognostic value .

The table below summarizes the relationship between MYSM1 expression and clinical parameters in CRC:

These findings suggest that MYSM1 suppresses CRC progression, potentially through epigenetic regulation of miR-200 family members and CDH1 and by inhibiting PI3K/AKT signaling .

Cardiovascular Research

Recent research using MYSM1 antibodies has uncovered MYSM1's role in myocardial ischemia/reperfusion (I/R) injury. A 2025 study identified a MYSM1-STAT1 axis in regulating myocardial I/R injury, positioning MYSM1 as a potential pharmacological target .

Western blot analysis using MYSM1 antibodies demonstrated that MYSM1 expression positively correlates with myocardial I/R injury. Mechanistically, MYSM1 was shown to mediate K63-linked deubiquitination and stabilization of STAT1 at position K379 via its MPN metalloprotease domain, initiating the expression of necroptosis-related genes .

Immunofluorescence staining with MYSM1 antibodies revealed that MYSM1 was predominantly increased in α-actinin+ cardiomyocytes, but not in vimentin+ fibroblasts and F4/80+ infiltrated macrophages, indicating cell-type specific expression during cardiac injury .

MYSM1 as a Biomarker in Cancer

Research using MYSM1 antibodies has revealed the potential of MYSM1 as a biomarker in cancer diagnosis and prognosis. Studies have shown that MYSM1 expression levels correlate with clinical outcomes in colorectal cancer, with higher expression associated with better prognosis .

Tissue microarray analysis with MYSM1 antibodies demonstrated that MYSM1 expression was inversely related to CRC stage, suggesting its potential utility as a prognostic marker . Similarly, in breast cancer, MYSM1 downregulation has been associated with more aggressive disease characteristics .

MYSM1 in Genetic Disorders

MYSM1 antibodies have been valuable in studying MYSM1-deficiency disorders in humans. Mutations in MYSM1 have been linked to a rare hereditary disorder characterized by leukopenia, anemia, and other hematopoietic and developmental abnormalities .

These antibodies enable the detection of aberrant MYSM1 expression or localization in patient samples, potentially aiding in diagnosis and understanding of disease mechanisms.

Western Blot Protocol

1. Sample Preparation:

   • Lyse cells in RIPA buffer containing protease inhibitors

   • Determine protein concentration using Bradford assay

2. SDS-PAGE:

   • Load 10-20 μg of protein per lane on 12% SDS-PAGE gel

   • Run at 100V until appropriate separation is achieved

3. Transfer:

   • Transfer proteins to nitrocellulose membrane at 100V for 1 hour

4. Blocking:

   • Block membrane with 5% non-fat dry milk in TBS-T for 1 hour at room temperature

5. Primary Antibody:

   • Dilute MYSM1 antibody 1:1000 in 5% milk/TBS-T

   • Incubate overnight at 4°C with gentle shaking

6. Washing:

   • Wash membrane 4 times for 5 minutes each with TBS-T

7. Secondary Antibody:

   • Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:20000) for 1 hour at room temperature

8. Detection:

   • Apply ECL substrate and image using chemiluminescence detection system

   • Expected band size: 95-110 kDa

Immunoprecipitation Protocol

1. Lysate Preparation:

   • Lyse 1x10^7 cells in 1 ml IP lysis buffer containing protease inhibitors

   • Centrifuge at 14,000g for 10 minutes at 4°C to clear lysate

   • Save 50 μl as input control

2. Antibody Binding:

   • Add 2-5 μg of MYSM1 antibody to 500 μl lysate

   • Incubate overnight at 4°C with rotation

3. Protein Capture:

   • Add 40 μl Protein A+G agarose beads

   • Incubate for 4 hours at 4°C with rotation

4. Washing:

   • Wash beads 3 times with IP lysis buffer

   • Centrifuge at 3,000g for 1 minute between washes

5. Elution and Analysis:

   • Resuspend beads in 30 μl 2X SDS sample buffer

   • Heat at 95°C for 5 minutes

   • Analyze by Western blot as described above

Co-Immunoprecipitation Protocol for Protein Interaction Studies

Co-immunoprecipitation protocols have been essential for investigating MYSM1's interactions with other proteins, such as STAT1, JAK1, and SHP2. A typical protocol includes:

1. Transfect cells with expression vectors (e.g., Flag-MYSM1)

2. Prepare cell lysates using IP lysis buffer

3. Immunoprecipitate with anti-Flag antibody or anti-Flag beads

4. Wash protein-bead mixtures three times with IP lysis buffer

5. Analyze by Western blot for both MYSM1 and potential interacting proteins

Future Perspectives and Emerging Applications

The development of more specific and sensitive MYSM1 antibodies continues to expand research possibilities. Emerging applications include:

1. Single-cell analysis: Next-generation MYSM1 antibodies compatible with single-cell proteomics will enable more detailed analysis of MYSM1 expression at the individual cell level.

2. Therapeutic monitoring: As MYSM1-targeted therapies emerge, antibodies will be essential for monitoring treatment efficacy and target engagement.

3. Multiplex imaging: Integration of MYSM1 antibodies into multiplex imaging platforms will facilitate comprehensive analysis of MYSM1 in complex tissue microenvironments.

4. CRISPR-Cas9 validation: MYSM1 antibodies will be crucial for validating CRISPR-Cas9 gene editing approaches targeting MYSM1 for functional studies.

Recent findings suggesting MYSM1's role in cardiovascular disease and cancer highlight the expanding scope of MYSM1 antibody applications beyond hematopoietic and immune system research .