MARCKSL1 Antibody

What is MARCKSL1 and why is it significant in research?

MARCKSL1, also known as MacMARCKS, F52, MLP, or MRP, is a member of the MARCKS family of protein kinase C substrates. It plays crucial roles in cellular signaling and is primarily localized to the plasma membrane . The protein functions as a key regulator of cell motility and has been implicated in cancer progression and metastasis .

MARCKSL1 is significant in research because:

• It interacts with F-actin to promote cancer cell invasion and migration

• It mediates invadopodia formation and extracellular matrix (ECM) degradation

• It functions in many aspects of cell physiology including integrin activation, cell spreading, and phagocytosis

• It has been associated with progression in multiple cancer types including esophageal squamous cell carcinoma and lung adenocarcinoma

What are the main applications for MARCKSL1 antibodies?

MARCKSL1 antibodies can be utilized in various experimental techniques:

• Western Blotting (WB): Most commercial MARCKSL1 antibodies are validated for WB at dilutions ranging from 1:500-1:2000 , with detected molecular weights typically between 42-60 kDa despite a calculated molecular weight of approximately 20 kDa .

• Immunohistochemistry (IHC): Used to examine MARCKSL1 expression in tissue samples, including patient tumor samples and tissue microarrays, at dilutions of approximately 1:50-1:100 .

• Immunofluorescence (IF): For colocalization studies examining MARCKSL1 interaction with other proteins like F-actin and cortactin, particularly in studies of invadopodia formation .

• ELISA: For quantitative detection of MARCKSL1, typically at dilutions of 1:5000-1:10000 .

• Immunoprecipitation (IP): For isolating MARCKSL1 and its binding partners to study protein-protein interactions .

What factors should be considered when selecting a MARCKSL1 antibody?

When selecting a MARCKSL1 antibody, researchers should consider:

• Target species reactivity: Different antibodies show varying reactivity with human, mouse, rat, and other species samples . Ensure the antibody is validated for your species of interest.

• Antibody type: Both polyclonal and monoclonal antibodies are available. Polyclonal antibodies offer higher sensitivity but potentially lower specificity, while monoclonal antibodies provide consistent results with high specificity .

• Host species: Consider the host species (rabbit, mouse) in relation to secondary antibodies and other reagents in your experimental design .

• Targeted region: Some antibodies target specific amino acid regions (e.g., AA 107-195, AA 132-160, C-terminal or N-terminal regions) , which may be important depending on your research questions.

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, IF, etc.) .

• Purification method: Antigen affinity-purified antibodies typically provide higher specificity .

How can MARCKSL1 antibodies be effectively used in cancer research?

MARCKSL1 antibodies have been instrumental in cancer research, particularly in studies examining:

• Expression profiling: MARCKSL1 is significantly overexpressed in several cancers. In lung adenocarcinoma, its elevated expression correlates with poor survival rates . Antibodies can be used to compare expression levels between tumor and normal tissues through IHC or WB techniques.

• Metastasis mechanisms: MARCKSL1 promotes cancer cell migration and invasion. Immunofluorescence studies using MARCKSL1 antibodies have demonstrated that overexpression of MARCKSL1 increases the colocalization of F-actin and cortactin at the frontier edge of migrating cells, enhancing invadopodia formation and ECM degradation .

• Prognostic studies: Though results have been mixed, MARCKSL1 antibodies have been used in validating MARCKSL1 as a potential prognostic marker in lymph node-negative breast cancer patients .

• Mechanistic investigations: When combined with knockdown studies, MARCKSL1 antibodies can help elucidate the mechanisms by which MARCKSL1 influences cancer progression. For example, silencing MARCKSL1 decreased the expression of EMT-associated proteins (E-cadherin, N-cadherin, vimentin) and decreased AKT phosphorylation in lung adenocarcinoma cells .

What technical challenges exist when using MARCKSL1 antibodies in colocalization studies?

When using MARCKSL1 antibodies for colocalization studies, particularly with F-actin, researchers should address several technical challenges:

• Fixation methods: The choice of fixation can affect both F-actin preservation and MARCKSL1 antibody epitope accessibility. Paraformaldehyde fixation (typically 4%) is often suitable for both.

• Signal-to-noise ratio: MARCKSL1 localizes to the membrane, where F-actin signals can also be intense. Optimizing antibody dilutions and using appropriate blocking solutions are critical to minimize background.

• Cross-reactivity concerns: Since MARCKSL1 shares approximately 50% amino acid homology with MARCKS , antibody specificity must be carefully validated, particularly in studies where both proteins are expressed.

• Appropriate controls: Studies examining the colocalization of MARCKSL1 with F-actin should include appropriate controls, such as MARCKSL1 knockdown cells, to confirm specificity of staining patterns .

• Advanced imaging techniques: Super-resolution microscopy may be necessary to accurately assess colocalization at subcellular structures like invadopodia, which are typically smaller than the resolution limit of conventional light microscopy.

How can MARCKSL1 and MARCKS antibodies be used to distinguish between their potentially redundant functions?

MARCKSL1 and MARCKS share structural similarities and may have partially redundant functions, making it challenging to study their individual contributions. Research strategies include:

• Differential expression analysis: Using specific antibodies against each protein to map their expression patterns in various tissues and developmental stages. For example, in tadpole spinal cord, Marcks and Marcksl1 show distinct but overlapping expression patterns, with Marcksl1 showing stronger immunostaining in the spinal cord but weaker staining in the meninges compared to Marcks .

• Knockout/knockdown comparisons: Using antibodies to validate the efficiency of gene silencing in single and double knockdown/knockout models to assess phenotypic differences. This approach revealed that Marcks and Marcksl1 have partly redundant functions in spinal cord development .

• Post-translational modification analysis: Specialized antibodies that detect specific phosphorylation states can reveal differences in regulation between MARCKSL1 and MARCKS.

• Protein-protein interaction studies: Immunoprecipitation with specific antibodies followed by mass spectrometry can identify unique binding partners for each protein, helping to distinguish their functions.

What are the methodological considerations when using MARCKSL1 antibodies in knockdown validation studies?

When using siRNA or CRISPR to knock down MARCKSL1, antibody-based validation is critical:

• Timepoint considerations: MARCKSL1 protein levels should be assessed at optimal timepoints after transfection. Studies show effective knockdown can be detected 48-72 hours post-transfection in lung adenocarcinoma cell lines .

• Quantification methods: Western blot analysis should include appropriate loading controls and densitometric quantification. Studies have shown that MARCKSL1-specific siRNAs can achieve significant protein knockdown (>70%) in A549 and H1975 lung cancer cell lines .

• Functional validation: Beyond simply confirming protein reduction, researchers should assess functional outcomes like:

  • Reduced cell migration in wound-healing assays

  • Decreased invasion in Transwell assays

  • Altered expression of downstream targets like EMT markers

• Antibody specificity: The disappearance of the specific MARCKSL1 band after knockdown serves as an additional validation of antibody specificity. Studies in CRISPR-modified tadpoles showed almost no Marcksl1 immunostaining in tissues where the gene was knocked out .

How should researchers interpret discrepancies between MARCKSL1's calculated and observed molecular weights?

MARCKSL1 has a calculated molecular weight of approximately 20 kDa but typically appears at 42-60 kDa on SDS-PAGE . When encountering this discrepancy:

• Verification approaches:

  • Use positive control lysates known to express MARCKSL1 (e.g., mouse brain tissue, 293T cells, SH-SY5Y cells)

  • Include knockdown/knockout samples as negative controls

  • Consider multiple antibodies targeting different regions of the protein

• Possible explanations:

  • Post-translational modifications, particularly phosphorylation, can significantly alter migration

  • The protein's high alanine content can affect SDS binding and electrophoretic mobility

  • Detergent-resistant membrane association may affect solubilization and migration

• Experimental considerations:

  • Different lysis buffers may reveal different banding patterns

  • Include appropriate molecular weight markers spanning the 20-60 kDa range

  • Consider phosphatase treatment to determine if phosphorylation contributes to the observed shift

What approaches can resolve contradictory findings regarding MARCKSL1's prognostic value in different cancer studies?

Research has produced conflicting results regarding MARCKSL1's prognostic value. For example, while high expression was associated with poor survival in lung adenocarcinoma , a validation study in breast cancer failed to confirm its prognostic value . To address such inconsistencies:

• Methodological standardization:

  • Use consistent antibody dilutions and scoring systems across studies

  • Standardize cutoff values for defining "high" versus "low" expression

  • Ensure comparable patient cohorts regarding treatment history

• Context-dependent analysis:

  • Consider changes in treatment protocols between discovery and validation cohorts

  • Analyze subgroups based on other molecular markers or clinical parameters

  • Account for differences in sample collection and processing methods

• Multivariate approaches:

  • Always include established prognostic factors in multivariate analyses

  • Consider MARCKSL1 in combination with other markers rather than in isolation

  • Report hazard ratios with confidence intervals rather than just p-values

What are the optimal experimental controls when using MARCKSL1 antibodies in fluorescence microscopy?

When performing immunofluorescence experiments with MARCKSL1 antibodies:

• Essential negative controls:

  • Secondary antibody only (omitting primary MARCKSL1 antibody)

  • MARCKSL1 knockdown or knockout samples

  • Non-immune IgG from the same species as the primary antibody

• Positive controls:

  • Cells known to express high levels of MARCKSL1 (e.g., SH-SY5Y neuroblastoma cells)

  • Co-staining with markers of known MARCKSL1 functions (e.g., F-actin for invadopodia studies)

• Technical considerations:

  • Include a nuclear counterstain to identify individual cells

  • Acquire z-stacks when examining membrane localization

  • Include scale bars and consistent exposure settings between experimental conditions

  • Consider super-resolution techniques for detailed localization studies

How can MARCKSL1 antibodies be used in neurodevelopmental research?

Recent studies have revealed important roles for MARCKSL1 in neural development, particularly in the spinal cord:

• Developmental expression profiling:

  • MARCKSL1 antibodies can map expression patterns during embryonic and postnatal development

  • Studies have shown that Marcksl1 is expressed at low levels in tadpole spinal cord but becomes upregulated after spinal cord injury

• Functional studies in neural regeneration:

  • MARCKSL1 and MARCKS have partly redundant functions required for normal neurite formation

  • Immunostaining with MARCKSL1 antibodies has shown increased expression in cells that infiltrate spinal cord injury sites

• Experimental approaches:

  • Combine MARCKSL1 immunostaining with markers of neural progenitors, mature neurons, and glial cells

  • Use CRISPR-modified models with MARCKSL1 knockout to assess developmental outcomes

  • Perform time-course analyses following injury to track MARCKSL1 expression changes

What factors influence the choice between monoclonal and polyclonal MARCKSL1 antibodies for different applications?

The decision between monoclonal and polyclonal MARCKSL1 antibodies depends on several factors:

• Monoclonal antibodies (e.g., MARCKSL1 Antibody K53, mouse monoclonal ):

  • Advantages: Consistent lot-to-lot reproducibility; higher specificity for a single epitope; reduced background in certain applications

  • Optimal applications: Diagnostic IHC; flow cytometry; applications requiring absolute specificity

  • Limitations: May be more sensitive to epitope masking by fixation; potentially lower sensitivity

• Polyclonal antibodies (e.g., MARCKSL1 Polyclonal Antibody CAB24471 ):

  • Advantages: Recognition of multiple epitopes increasing detection sensitivity; robustness against protein denaturation

  • Optimal applications: Western blotting for low-abundance proteins; IHC of fixed tissues

  • Limitations: Batch-to-batch variation; potential for higher background

• Application-specific considerations:

  • For detecting post-translational modifications: Monoclonal antibodies raised against specific modified epitopes

  • For colocalization studies: Consider using monoclonal antibodies from different species for MARCKSL1 and other targets

  • For quantitative analyses: Consistent lots of the same antibody should be used throughout the study

How should researchers address cellular localization discrepancies in MARCKSL1 antibody-based studies?

MARCKSL1 primarily localizes to the plasma membrane, but studies may show different localization patterns. To address these inconsistencies:

• Subcellular fractionation validation:

  • Perform cellular fractionation followed by Western blotting to biochemically verify MARCKSL1 distribution

  • Include markers for different cellular compartments (membrane, cytosol, nucleus)

• Fixation and permeabilization optimization:

  • Compare different fixation methods (paraformaldehyde, methanol, acetone)

  • Optimize permeabilization conditions to maintain membrane structure while allowing antibody access

• Context-dependent localization:

  • Consider cellular activation state, as PKC activation may alter MARCKSL1 localization

  • Examine localization during different phases of cell migration or cell cycle

  • Assess potential redistribution in response to experimental treatments

• Technical approaches:

  • Use live-cell imaging with fluorescently-tagged MARCKSL1 as complementary evidence

  • Consider super-resolution microscopy for precise localization studies

  • Employ proximity ligation assays to confirm interactions with known binding partners