MEMO1 Antibody

What is MEMO1 protein and what are its primary functions?

MEMO1 is a 297-amino acid protein that plays crucial roles in regulating cell migration by modulating extracellular chemotactic signals directed at the microtubule cytoskeleton . The protein's name derives from its function as a mediator of ERBB2-driven cell motility 1. MEMO1 mediates ERBB2 signaling, which is essential for the migration of breast carcinoma cells, making it particularly significant in cancer research . Recent research has identified MEMO1 as an iron-binding protein that modulates iron homeostasis in cancer cells, adding another dimension to its biological importance .

In cancer contexts, MEMO1 supports the ability of breast tumor cells to invade surrounding tissues, leading to metastasis . Knockdown of MEMO1 expression reduces breast cancer cell migration in culture and significantly suppresses lung metastasis in xenograft models . Clinical studies have shown a strong correlation between increased MEMO1 expression and reduced patient survival in cancer patients .

Beyond cancer, MEMO1 has been identified as a novel regulator of magnesium homeostasis and systemic calcification propensity, particularly through its effects on regulating the expression of magnesium channels in the kidney . This multifaceted protein continues to reveal new biological functions as research progresses.

What types of MEMO1 antibodies are available for research?

Researchers have access to several types of MEMO1 antibodies for different experimental applications:

• Monoclonal antibodies: Such as MEMO1 Antibody (AT1E9), a mouse monoclonal IgG1 kappa light chain antibody that detects MEMO1 protein across multiple species . These antibodies offer high specificity and consistency between lots.

• Polyclonal antibodies: Including rabbit polyclonal antibodies targeting specific regions of MEMO1, such as the C-terminal region . These antibodies can provide higher sensitivity by recognizing multiple epitopes.

• Antibody conjugates and bundles: Some suppliers offer MEMO1 antibodies bundled with secondary detection reagents, such as:

  • m-IgG Fc BP-HRP Bundle for enhanced western blotting applications

  • m-IgGκ BP-HRP Bundle for sensitive detection systems

These antibodies are available in various formats (100 μg/ml is common) and can be used in multiple experimental techniques, including western blotting, immunoprecipitation, immunofluorescence, flow cytometry, and ELISA .

What species reactivity can be expected with common MEMO1 antibodies?

MEMO1 antibodies show cross-reactivity across multiple species due to the conserved nature of the protein. Based on available information, common reactivity profiles include:

The rabbit polyclonal antibody targeting the C-terminal region shows particularly broad reactivity across species . The calculated molecular weight of MEMO1 is approximately 32 kDa, which serves as an important reference point when validating antibody specificity .

When selecting an antibody for your research, it's crucial to verify the specific reactivity of your chosen antibody with the supplier, especially if working with less common model organisms.

What are the key applications for MEMO1 antibodies in research?

MEMO1 antibodies support multiple research applications, making them versatile tools for investigating MEMO1's role in normal and pathological processes:

These applications are particularly valuable in cancer research, where MEMO1's roles in cell migration, iron homeostasis, and metastasis make it an important target for investigation . For iron homeostasis studies, these techniques can reveal how MEMO1 interacts with mitochondrial iron transporters and affects cellular responses to iron availability .

How should I optimize western blotting protocols for MEMO1 detection?

Optimizing western blotting for MEMO1 detection requires attention to several key parameters:

For sample preparation, use RIPA or NP-40 lysis buffers supplemented with protease inhibitors to prevent degradation of MEMO1 protein . Include phosphatase inhibitors if studying phosphorylation states or signaling pathways. Remember that the calculated molecular weight of MEMO1 is approximately 32 kDa, which helps identify the correct band .

When performing electrophoresis and transfer, use 10-12% polyacrylamide gels for optimal resolution of proteins in this size range. Ensure complete transfer to PVDF or nitrocellulose membranes (30V overnight or 100V for 1 hour) to avoid inconsistent results.

For antibody incubation, primary antibody dilutions typically range from 1:500 to 1:2000 in 5% BSA or milk, though this should be optimized for your specific antibody . Incubate overnight at 4°C with gentle agitation for best results. Secondary antibody dilution should be around 1:5000 to 1:10000 for 1 hour at room temperature.

Always include positive controls, such as lysates from cell lines known to express MEMO1 (e.g., MDA-MB-231 breast cancer cells, which have been used in MEMO1 research) . For validating specificity, MEMO1 knockout samples provide excellent negative controls.

What are the recommended immunofluorescence protocols for MEMO1 antibodies?

Successful immunofluorescence staining for MEMO1 requires careful attention to fixation, permeabilization, and antibody incubation conditions:

For fixation and permeabilization, fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1-0.5% Triton X-100 for 10 minutes. Alternative methods include methanol fixation at -20°C for 10 minutes, which combines fixation and permeabilization in one step.

Block with 5% normal serum (from the species of the secondary antibody) in PBS with 0.1% Tween-20 for 1 hour to minimize non-specific binding. For antibody incubation, dilute primary MEMO1 antibody 1:100 to 1:500 in blocking buffer and incubate overnight at 4°C in a humidified chamber .

For co-localization studies investigating MEMO1's role in mitochondrial function, consider co-staining with mitochondrial markers such as GRP75/HSPA9 . This approach has revealed important insights about MEMO1's involvement in mitochondrial morphology and function, particularly under iron-depleted conditions .

In research contexts, this methodology has demonstrated that MEMO1 knockout cells exposed to iron chelators (DFX) show abnormal perinuclear mitochondrial clustering, while wild-type cells maintain normal mitochondrial distribution . This finding highlights MEMO1's role in maintaining mitochondrial integrity under iron-limited conditions.

How can I verify the specificity of MEMO1 antibodies?

Verifying antibody specificity is crucial for generating reliable research data. For MEMO1 antibodies, consider these comprehensive approaches:

Genetic controls provide the gold standard for antibody validation. Use MEMO1 knockout cell lines (such as CRISPR/Cas9-generated lines) as negative controls to confirm absence of signal. Similarly, employ siRNA or shRNA knockdown of MEMO1 to demonstrate signal reduction proportional to the knockdown efficiency . Overexpression systems with tagged MEMO1 constructs can further confirm signal increase and specificity.

Analytical approaches should include peptide competition assays to block specific binding and confirmation of the expected molecular weight by western blotting (approximately 32 kDa) . Validate that subcellular localization patterns match known MEMO1 distribution.

For independent validation, compare results across different detection methods (e.g., immunofluorescence versus western blotting) to ensure consistent findings. When possible, correlate protein detection with mRNA expression data to provide orthogonal validation.

Researchers studying MEMO1's role in iron homeostasis and mitochondrial function have utilized MEMO1 knockout MDA-MB-231 cells as negative controls, which helped establish the specificity of antibody staining patterns in mitochondrial co-localization studies . This approach enabled reliable detection of MEMO1-dependent changes in mitochondrial morphology under iron-depleted conditions.

What considerations are important when using MEMO1 antibodies for immunoprecipitation?

Immunoprecipitation (IP) of MEMO1 can yield valuable insights into protein interactions but requires optimization of several parameters:

For lysis conditions, use gentle lysis buffers (e.g., NP-40 or CHAPS-based) to preserve protein-protein interactions, as harsh detergents may disrupt important complexes . Include protease and phosphatase inhibitors to prevent degradation and maintain post-translational modifications that may be critical for interactions.

Antibody selection is crucial for successful IP experiments. Choose antibodies specifically validated for IP applications . Monoclonal antibodies often provide cleaner results with less background, but polyclonal antibodies may capture more diverse complexes due to recognition of multiple epitopes.

When studying MEMO1's interactome, consider the specific biological context. For investigating MEMO1's interaction with ERBB2, use mild detergent conditions to preserve receptor-associated complexes . When studying iron-related interactions, be aware that metal chelators might disrupt important binding events. For mitochondrial interactions, such as with SLC25A28 (mitoferrin-2), mitochondrial isolation prior to IP may provide cleaner results .

Always include appropriate controls: IgG isotype control to identify non-specific binding, input sample (pre-IP lysate) to confirm target protein presence, and MEMO1 knockout/knockdown samples as negative controls for specificity validation.

How can MEMO1 antibodies be used to study iron homeostasis in cancer cells?

Recent research has revealed MEMO1 as an iron-binding protein that modulates iron homeostasis in cancer cells . MEMO1 antibodies can be leveraged to investigate this function through several sophisticated approaches:

Immunofluorescence co-staining of MEMO1 with mitochondrial iron transporters (especially SLC25A28/mitoferrin-2) can reveal spatial relationships and potential functional interactions . This approach has demonstrated that MEMO1 knockout cells exposed to iron chelator DFX (1μM) exhibit abnormal perinuclear mitochondrial clustering, while wild-type cells maintain normal mitochondrial distribution . This finding suggests that MEMO1 plays a critical role in maintaining mitochondrial integrity under iron-limited conditions.

Fractionation studies using MEMO1 antibodies can track subcellular localization changes in response to iron availability. Western blotting of mitochondrial, cytoplasmic, and nuclear fractions helps monitor MEMO1 distribution across cellular compartments under different iron conditions.

Research using these methods has revealed that MEMO1 knockout cells show decreased cytotoxicity to RSL3 (a ferroptosis-inducing agent that inhibits GPX4), suggesting that high-MEMO1 cells are more sensitive to ferroptosis . This links MEMO1's iron-binding function to cellular sensitivity to iron-dependent cell death pathways.

These approaches collectively help elucidate how MEMO1 contributes to iron homeostasis and potentially identify new therapeutic targets in cancers with high MEMO1 expression .

What methods can be used to investigate MEMO1's role in cancer cell migration?

MEMO1 was originally identified for its role in cancer cell migration and metastasis . Researchers can use MEMO1 antibodies in several experimental approaches to investigate this critical function:

Transwell migration assays comparing wild-type and MEMO1 knockdown/knockout cells provide quantitative measurements of migration capacity. Wound healing assays with immunofluorescence staining for MEMO1 at the leading edge reveal dynamic localization during collective cell migration. Three-dimensional invasion assays in extracellular matrix better recapitulate in vivo conditions for assessing invasive capacity.

For cytoskeletal studies, co-immunoprecipitation of MEMO1 with cytoskeletal proteins can identify direct interactions with the migration machinery . Immunofluorescence co-localization of MEMO1 with microtubules helps visualize how MEMO1 mediates ERBB2 receptor signals to the cytoskeleton, affecting cell motility .

Published research has demonstrated that knockdown of MEMO1 expression reduces breast cancer cell migration in culture and significantly suppresses lung metastasis in xenograft models . Additionally, retrospective analysis of resected tumors showed a strong correlation between increased MEMO1 expression and reduced patient survival . These findings highlight the clinical relevance of MEMO1's role in cancer cell migration and metastasis.

How can MEMO1 antibodies help explore the relationship between MEMO1 and ERBB2 signaling?

The interaction between MEMO1 and ERBB2 (HER2) is central to understanding MEMO1's role in cancer, particularly in breast cancer . MEMO1 antibodies can be utilized in several ways to investigate this relationship:

Co-immunoprecipitation studies using MEMO1 antibodies to pull down protein complexes and probe for ERBB2 can directly demonstrate physical interactions between these proteins . Reverse co-IP with ERBB2 antibodies can further confirm this association. These experiments can reveal how the interactions change with ERBB2 activation or inhibition by therapeutic agents.

Proximity ligation assays provide a powerful method to visualize and quantify MEMO1-ERBB2 interactions in situ, offering spatial information about where in the cell these interactions occur. This technique can assess how these interactions change with receptor activation or drug treatment.

Research has established that the interaction with ERBB2 gave MEMO1 its name (mediator of ERBB2-driven cell motility 1) . This interaction is proposed to relay the activation of ERBB receptor heterodimers to the microtubule cytoskeleton, inducing the growth of lamellipodia and enabling cancer cell migration . Understanding this relationship has significant implications for developing strategies to inhibit cancer metastasis, particularly in HER2-positive cancers.

What approaches are recommended for studying MEMO1's impact on mitochondrial function?

Recent research has revealed MEMO1's involvement in mitochondrial function, particularly in relation to iron homeostasis . These approaches can help investigate this relationship:

Immunofluorescence co-staining of MEMO1 with mitochondrial markers like GRP75 (HSPA9) enables visualization of MEMO1's association with mitochondria . Confocal microscopy can be used to assess mitochondrial morphology in wild-type versus MEMO1 knockout cells under normal conditions and following application of mitochondrial stressors.

For functional assessments, measure mitochondrial membrane potential using dyes like MitoTracker CM-H2Ros in cells with varying MEMO1 expression . This approach has revealed that cells with MEMO1 knockout show abnormal perinuclear mitochondrial clustering when treated with iron chelator DFX, while wild-type cells maintain normal mitochondrial distribution .

Studies of ferroptosis sensitivity have shown that MEMO1 knockout results in decreased cytotoxicity to RSL3 in breast cancer cells, indicating that high-MEMO1 cells are more sensitive to this iron-dependent cell death pathway . Under basal iron conditions, the GPX4 level (a key anti-ferroptotic enzyme) in high-MEMO1 cells was approximately twice higher compared to knockout cells, but this difference disappeared under elevated iron conditions .

These approaches collectively help elucidate MEMO1's role in maintaining mitochondrial integrity and function, particularly in relation to iron metabolism and ferroptosis sensitivity.

Why might I observe variable MEMO1 expression across different cancer cell lines?

Variability in MEMO1 expression across cancer cell lines is common and can be attributed to several biological and technical factors:

From a biological perspective, tissue of origin differences naturally affect MEMO1 expression patterns. MEMO1 is located on human chromosome 2, a region containing over 1,400 genes and accounting for nearly 8% of the human genome . Genetic alterations affecting this chromosome may influence MEMO1 expression. Additionally, MEMO1 interacts with the extranuclear estrogen receptor, potentially affecting its expression in hormone-responsive cancers .

Experimental factors can also contribute to variability. Culture conditions, including growth factors in media, may influence MEMO1 expression. Cell confluency and passage number can affect expression profiles, making standardization important for comparative studies.

Research has shown that MEMO1 is overexpressed in many types of cancer and its increased expression correlates with reduced patient survival . In breast cancer specifically, MEMO1 expression has been linked to metastatic potential, with knockdown of MEMO1 significantly suppressing lung metastasis in xenograft models .

To address this variability, normalize MEMO1 expression to multiple housekeeping genes, compare expression at both protein and mRNA levels, and maintain consistent experimental conditions across comparative studies.

How can I address non-specific binding issues with MEMO1 antibodies?

Non-specific binding is a common challenge when working with antibodies. For MEMO1 antibodies, consider these optimization strategies:

Begin by titrating antibody concentrations to find the optimal signal-to-noise ratio. Testing different blocking agents (BSA, milk, normal serum) can significantly reduce background. Commercial MEMO1 antibodies typically come in formats like 100 μg/ml, and proper dilution is critical for specificity .

For western blotting applications, optimize transfer conditions to ensure complete protein transfer, particularly important for the 32 kDa MEMO1 protein . Consider membrane blocking time and temperature, and use freshly prepared buffers and reagents to minimize artifacts.

Buffer modifications can significantly improve specificity. Adding 0.1-0.5% Tween-20 reduces hydrophobic interactions, while including 150-300mM NaCl helps minimize ionic interactions. For immunofluorescence, proper permeabilization with 0.1-0.5% Triton X-100 is essential for antibody access to intracellular MEMO1.

Control experiments are crucial for validating specificity. Include MEMO1 knockout/knockdown samples as negative controls whenever possible . Use isotype control antibodies at the same concentration to identify any non-specific binding related to the antibody class rather than its specificity for MEMO1.

What controls are essential when analyzing MEMO1 knockdown/knockout experiments?

For genetic controls, use multiple independent knockdown/knockout clones to account for clonal variation. In published studies, MEMO1 knockout MDA-MB-231 and A-375 cell lines have been successfully used to investigate MEMO1 functions . Include non-targeting siRNA/sgRNA controls with similar chemical modifications to account for any off-target effects of the knockdown methodology.

Expression validation should confirm knockdown/knockout at both protein level (using MEMO1 antibodies) and mRNA level (RT-qPCR). This multi-level validation ensures complete loss of MEMO1 function rather than just reduction in protein levels.

For iron-related studies, include both iron chelator (e.g., DFX) and iron supplementation conditions as functional controls . Research has shown that 1 μM DFX (a high subtoxic concentration) reveals differential responses between wild-type and MEMO1 knockout cells in terms of mitochondrial morphology .

When studying ferroptosis, incorporate known ferroptosis inducers (e.g., RSL3) and inhibitors as functional controls . These controls help establish whether observed phenotypes are specifically related to MEMO1's role in iron homeostasis and ferroptosis sensitivity.

How should I reconcile conflicting data regarding MEMO1 function in different cell types?

Conflicting results across different cellular contexts are common in molecular biology research. For MEMO1, consider these approaches to reconcile seemingly contradictory findings:

Begin with systematic analysis of experimental differences. Document the exact cell lines used in conflicting studies, their genetic backgrounds, culture conditions, and methodological differences. For example, MEMO1's roles have been studied in various cancer cell lines including MDA-MB-231 (breast cancer) and A-375 (melanoma) .

Consider MEMO1's multiple biological functions. While originally characterized for its role in ERBB2-mediated cell migration in breast cancer cells , MEMO1 also functions as an iron-binding protein that modulates iron homeostasis and regulates magnesium homeostasis . Different cell types may emphasize different aspects of MEMO1's functional repertoire.

Investigate context-dependent protein interactions. MEMO1 interacts with different signaling pathways that may predominate in different cell types. Its interactions with ERBB2, the insulin receptor substrate protein 1 pathway, and the extranuclear estrogen receptor may vary in importance depending on cell type .

Research has shown that MEMO1's impact on mitochondria is particularly evident under iron-depleted conditions . Wild-type cells maintain normal mitochondrial distribution when treated with iron chelator DFX, while MEMO1 knockout cells show perinuclear mitochondrial clustering . This suggests that MEMO1's functions may become more critical under specific cellular stresses, which may not be apparent under all experimental conditions.

How can MEMO1 antibodies be utilized in studying magnesium homeostasis?

Recent research has identified MEMO1 as a novel regulator of magnesium homeostasis . MEMO1 antibodies can be instrumental in exploring this emerging function:

Immunohistochemistry of kidney tissues using MEMO1 antibodies can examine expression patterns in relation to magnesium transport. This approach is particularly relevant given that kidney-specific MEMO1 knockout (kKO) mice display higher magnesemia and increased renal magnesium channel gene expression .

Co-localization studies with known magnesium channels and transporters can reveal potential functional interactions. Immunoprecipitation experiments using MEMO1 antibodies can identify interactions with magnesium regulatory proteins, providing mechanistic insights into how MEMO1 modulates magnesium homeostasis.

Research has demonstrated that both conditional MEMO1 knockout (cKO) and kidney-specific MEMO1 knockout (kKO) mice exhibit elevated serum magnesium levels . These models reveal that MEMO1 regulates magnesium homeostasis and systemic calcification propensity by controlling the expression of the main magnesium channels . This finding connects MEMO1 to broader physiological functions beyond its established roles in cancer.

Investigating MEMO1 expression in patients with magnesium metabolism disorders may reveal novel therapeutic targets. The link between MEMO1, magnesium homeostasis, and calcification propensity suggests potential applications in conditions involving altered mineral balance.

What are the newest methodologies for investigating MEMO1's role in ferroptosis?

Ferroptosis, a form of iron-dependent cell death, has recently been linked to MEMO1 function . These cutting-edge approaches can help investigate this connection:

Advanced cell death assays using real-time monitoring with time-lapse microscopy and fluorescent probes provide dynamic information about ferroptosis progression. Multiplexed cell death assays can distinguish ferroptosis from other cell death modes like apoptosis and necroptosis, essential for accurately characterizing MEMO1's specific effect on ferroptotic pathways.

Genetic engineering approaches including CRISPR/Cas9 screening can identify genes that modify MEMO1-dependent ferroptosis sensitivity. Generation of MEMO1 domain mutants can pinpoint regions critical for ferroptosis regulation, providing mechanistic insights into how MEMO1 influences this process.

Research has established that MEMO1 knockout results in decreased cytotoxicity to RSL3 (a ferroptosis inducer that inhibits GPX4) in breast cancer cells, indicating that high-MEMO1 cells are more sensitive to ferroptosis . Under basal iron conditions, GPX4 expression in high-MEMO1 cells was approximately twice higher compared to knockout cells, though this difference disappeared under elevated iron conditions .

These findings suggest that MEMO1 may serve as a biomarker for tumors particularly sensitive to therapies targeting iron metabolism in cancer cells . Understanding the mechanisms by which MEMO1 modulates ferroptosis sensitivity could lead to novel therapeutic strategies.

How might MEMO1 serve as a biomarker for tumors sensitive to iron metabolism-targeting therapies?

The emerging role of MEMO1 in iron homeostasis suggests potential as a biomarker for predicting response to therapies targeting iron metabolism :

Immunohistochemical staining of tumor tissue microarrays using MEMO1 antibodies can correlate expression with clinical outcomes. Development of quantitative MEMO1 detection methods in patient samples could facilitate biomarker development. Integrating MEMO1 expression data with other iron metabolism markers may provide a more comprehensive predictive profile.

Preclinical validation can be achieved by screening diverse cancer cell lines for correlation between MEMO1 expression and sensitivity to iron chelators. Xenograft models comparing therapy response in tumors with varying MEMO1 expression provide in vivo validation. Patient-derived organoids offer opportunities to test personalized approaches based on MEMO1 status.

Research has shown that high-MEMO1 cells exhibit increased sensitivity to ferroptosis inducers like RSL3 . MEMO1 overexpression may help maintain normal metabolism of cancer cells by increasing iron levels in mitochondria under hypoxic conditions . The structure and iron coordination mode of MEMO1 suggest its involvement in biosynthesis or processing of signaling molecules .

These findings suggest that MEMO1 may serve as a biomarker of tumors particularly sensitive to therapies targeting iron metabolism in the cell . GIs (genomic instabilities) of MEMO1 may be targeted to suppress metastasis in breast cancer and other malignancies with high-MEMO1 expression levels .

What are the current approaches to studying MEMO1's interaction with the mitochondrial iron transporter SLC25A28?

The interaction between MEMO1 and SLC25A28 (mitoferrin-2) represents an exciting research direction in understanding mitochondrial iron homeostasis :

Protein-protein interaction analysis using co-immunoprecipitation with MEMO1 antibodies followed by SLC25A28 detection can directly demonstrate physical association. Proximity ligation assays visualize interactions in situ, providing spatial information about where in the cell these interactions occur. These techniques can reveal how the MEMO1-SLC25A28 interaction changes under varying conditions.

Functional characterization through mitochondrial iron uptake assays in cells with MEMO1 knockout/knockdown can establish the functional significance of this interaction. Rescue experiments with wild-type versus interaction-deficient MEMO1 mutants help determine whether the interaction is essential for MEMO1's effects on mitochondrial iron homeostasis.

Research has shown that the decrease in mitochondrial iron in MEMO1 knockout cells correlates with interactions between MEMO1 and SLC25A28 . MEMO1 knockout cells treated with iron chelator DFX show abnormal perinuclear mitochondrial clustering, while wild-type cells maintain normal mitochondrial distribution . This suggests that MEMO1 plays a critical role in maintaining mitochondrial integrity under iron-limited conditions through its interaction with the mitochondrial iron transport machinery.

Understanding this interaction may provide insights into mitochondrial iron regulation in both normal physiology and disease states, potentially revealing new therapeutic opportunities for conditions involving disrupted iron homeostasis.