MYO1D Antibody

What is MYO1D and what are its main functions?

MYO1D is an unconventional myosin that functions as an actin-based motor protein with ATPase activity. It plays critical roles in multiple cellular processes including:

• Endosomal protein trafficking, especially in the transfer of cargo proteins from early to recycling endosomes

• Maintaining epithelial integrity and protecting against colitis

• Contributing to normal planar cell polarity in ciliated tracheal cells

• Supporting normal rotational polarity of cilia and coordinated ciliary movement

• Coupling cytoskeletal elements to lipid membranes in an ATP-dependent manner

In the enterocyte, MYO1D localizes to the basolateral membrane, brush border terminal web, and is enriched in puncta at the distal tips of microvilli . MYO1D is also expressed in mature oligodendrocytes and may be involved in the formation of myelin-like membrane sheets .

How do I select the appropriate MYO1D antibody for my experiment?

When selecting a MYO1D antibody, consider the following factors:

• Experimental application: Different antibodies perform better in specific applications. For example, some antibodies work well in Western blot but not in immunohistochemistry. According to available data, MYO1D antibodies have been validated for Western blot, ELISA, immunofluorescence, and immunohistochemistry .

• Species reactivity: Verify that the antibody recognizes MYO1D in your species of interest. Most commercial MYO1D antibodies react with human, mouse, and rat MYO1D .

• Epitope location: Different antibodies target distinct regions of MYO1D, which can affect localization patterns. For instance, the C13 antibody detects MYO1D at both the terminal web and microvillar tips, while K18 targets only the microvillar tips and H60 targets the terminal web and basolateral membrane .

• Clonality: Choose between monoclonal (more specific) and polyclonal (potentially higher sensitivity) based on your experimental needs. Both types are available for MYO1D .

• Validation data: Review images and validation methods provided by suppliers to ensure the antibody works in your application of interest .

What are the optimal conditions for using MYO1D antibodies in immunohistochemistry?

For optimal immunohistochemistry (IHC) results with MYO1D antibodies:

• Fixation: Most protocols use paraformaldehyde fixation (4% PFA) for tissue sections.

• Antigen retrieval: This may be necessary depending on the fixation method. Heat-induced epitope retrieval is commonly used.

• Blocking: Use 10% BSA in PBS for approximately 20 minutes to reduce background staining .

• Antibody dilution: Recommended dilutions vary by manufacturer but typically range from 1:50 to 1:300 for IHC applications. For example, Boster Bio recommends 1:100-1:300 dilution for their anti-MYO1D antibody , while Abbexa suggests 1:50-1:100 .

• Incubation conditions: Primary antibody incubation is typically performed for 1 hour at room temperature or overnight at 4°C .

• Detection system: Use an appropriate secondary antibody conjugated to a fluorophore (for immunofluorescence) or HRP (for chromogenic detection).

• Controls: Always include positive controls (tissues known to express MYO1D) and negative controls (omitting primary antibody).

How can I optimize Western blot protocols for MYO1D detection?

For successful Western blot detection of MYO1D:

• Sample preparation: Use appropriate lysis buffers that preserve protein integrity. RIPA buffer with protease inhibitors is commonly used.

• Protein loading: Load 25-50 μg of total protein per lane, as demonstrated in validation studies .

• Expected molecular weight: MYO1D has a calculated molecular weight of 116.2 kDa, though some researchers observe bands at approximately 72 kDa .

• Blocking: Use 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Antibody dilution: Typical dilutions range from 1:500 to 1:1000, though this varies by manufacturer. For example, ab70204 has been validated at 1:500 dilution .

• Washing: Perform thorough washing steps (3-5 times for 5-10 minutes each) with TBST between antibody incubations.

• Detection method: Both chemiluminescence and fluorescence-based detection systems work well with MYO1D antibodies.

• Positive control: Consider using lysates from MYO1D-transfected cells as a positive control, as demonstrated in validation studies .

How do I interpret different MYO1D localization patterns in my immunostaining results?

MYO1D demonstrates distinct localization patterns that can vary depending on:

• Cell/tissue type: In enterocytes, MYO1D localizes to the basolateral membrane, terminal web, and microvillar tips .

• Antibody epitope: Different antibodies targeting distinct C-terminal epitopes of MYO1D produce varied staining patterns. For example:

  • C13 antibody: Composite staining showing terminal web, basolateral membrane, and microvillar tip localization

  • K18 antibody: Primarily microvillar tip localization

  • H60 antibody: Terminal web and basolateral membrane localization

• Experimental conditions: Fixation, permeabilization, and antigen retrieval methods can affect epitope accessibility.

When interpreting localization results:

• Compare your findings with published patterns

• Consider using multiple antibodies targeting different epitopes to confirm localization

• Include co-staining with markers for cellular compartments (e.g., phalloidin for actin filaments)

• Be aware that MYO1D localization can change in response to experimental manipulations or disease states

What are common problems when using MYO1D antibodies and how can I troubleshoot them?

Common problems and troubleshooting approaches include:

• High background signal:

  • Increase blocking time or concentration (e.g., use 10% BSA instead of 5%)

  • Dilute primary antibody further

  • Include additional washing steps

  • Consider using different blocking agents (BSA, normal serum, commercial blockers)

• Weak or no signal:

  • Optimize antigen retrieval (if applicable)

  • Increase antibody concentration

  • Extend incubation time

  • Ensure sample preparation preserves the epitope

  • Check if protein expression varies by tissue region (e.g., MYO1D tip labeling is mainly observed along the distal half of intestinal villi)

• Unexpected band size in Western blot:

  • MYO1D has a calculated molecular weight of 116.2 kDa, but observed bands may differ

  • Consider post-translational modifications, proteolytic cleavage, or splice variants

  • Verify antibody specificity using positive and negative controls

• Inconsistent results between experiments:

  • Standardize fixation and processing protocols

  • Prepare fresh working solutions of antibodies

  • Be consistent with imaging parameters

  • Consider batch effects in antibody production

• Cross-reactivity issues:

  • Test the antibody on knockout/knockdown samples if available

  • Use peptide blocking to confirm specificity

  • Consider trying antibodies from different suppliers or targeting different epitopes

How can I study MYO1D dynamics in living cells?

To study MYO1D dynamics in living cells:

• Fluorescent protein fusion constructs:

  • Generate EGFP-tagged MYO1D constructs (full-length or domain-specific)

  • Ensure the tag doesn't interfere with function or localization

  • Express in appropriate cell models (e.g., CL4 cells for apical microvilli studies)

• FRAP (Fluorescence Recovery After Photobleaching) analysis:

  • FRAP studies have revealed important differences between MYO1D and related myosins

  • MYO1D shows higher dynamics compared to MYO1A, which has a larger immobile fraction

  • These differences in dynamics may explain differential localization patterns

• Truncation constructs for domain analysis:

  • Previous studies have used EGFP-Myo1d constructs to analyze domain requirements

  • Full-length Myo1d (aa 1-1006) and Myo1d-IQTH1 (aa 570-1006) localize to microvilli and basolateral membrane

  • Myo1d-TH1 (aa 748-1006) shows weak microvillar targeting

  • Both IQ and TH1 domains are necessary but not sufficient for proper targeting

• siRNA-mediated knockdown:

  • Use siRNA targeting MYO1D to study loss-of-function effects

  • Knockdown of MYO1D in oligodendrocytes reduces MBP-positive cells and disrupts myelin-like membrane sheets

How does MYO1D function differ from other class I myosins in epithelial cells?

MYO1D shows several functional and localization differences from other class I myosins:

• Localization patterns:

  • MYO1D localizes to the terminal web and microvillar tips in enterocytes

  • MYO1A distributes along the length of microvilli

  • In MYO1A knockout mice, MYO1D peptide counts increase approximately twofold, and MYO1D redistributes along the length of microvilli

• Domain requirements for targeting:

  • MYO1D requires both IQ and TH1 domains for proper targeting

  • MYO1A targeting is achieved through the TH1 domain alone

• Dynamics:

  • FRAP studies show that MYO1D has a smaller immobile fraction compared to MYO1A

  • These differences in dynamics may govern the differential localization of these closely related myosins

• Compensation mechanisms:

  • MYO1D may compensate for the loss of MYO1A in knockout mice

  • Proteomic analysis of MYO1A knockout brush borders shows a 131% increase in MYO1D peptide counts

• Functional roles:

  • MYO1D plays a role in protecting against colitis, as demonstrated by increased susceptibility to DSS-induced colitis in MYO1D mutant mice

  • MYO1D appears to couple cytoskeletal elements to lipid in an ATP-dependent manner

What approaches can be used to study the role of MYO1D in disease models?

Several approaches can be used to investigate MYO1D in disease contexts:

• Genetic models:

  • N-ethyl-N-nitrosourea (ENU) mutagenesis has generated MYO1D mutant mice that show increased susceptibility to DSS-induced colitis

  • CRISPR/Cas9 targeting has been used to validate MYO1D's role in protecting against colitis

  • Complementation testing can confirm the specificity of the phenotype

• Knockdown studies:

  • siRNA targeting MYO1D in oligodendrocytes showed that MYO1D is required for the formation of myelin-like membrane sheets

  • MYO1D knockdown decreased the number of MBP-positive cells and disrupted myelin-like membrane formation

• Bone marrow chimeras:

  • Bone marrow transplantation experiments can determine whether the disease phenotype depends on the hematopoietic or non-hematopoietic compartment

  • The colitis phenotype in MYO1D mutant mice is dependent on the non-hematopoietic compartment

• Biochemical interaction studies:

  • Investigate MYO1D's ATP-dependent coupling of cytoskeletal elements to lipid membranes

  • This function may be critical for maintaining epithelial integrity and protection against colitis

• Immunostaining in disease tissues:

  • Compare MYO1D expression and localization patterns between normal and disease tissues

  • Changes in expression or localization could provide insights into disease mechanisms

Can MYO1D antibodies be conjugated with biotin or other labels for specialized applications?

Yes, MYO1D antibodies can be conjugated with biotin or other labels, but several factors need consideration:

• Buffer requirements:

  • Antibodies should be free of BSA and sodium azide before conjugation

  • Buffer exchange to remove these components is possible through dialysis or desalting columns

  • PBS without additives is typically used as the final buffer

• Antibody concentration:

  • Concentration of 1 mg/ml is often optimal for conjugation reactions

  • Some commercial MYO1D antibodies are already provided at this concentration

• Storage after conjugation:

  • Conjugated antibodies can be stored at -20°C in small aliquots to avoid freeze-thaw cycles

  • Glycerol (50%) can be added as a cryoprotectant

• Validation after conjugation:

  • Conjugated antibodies should be validated to ensure that labeling hasn't affected binding specificity

  • Comparison with unconjugated antibody in parallel experiments is recommended

• Available commercial options:

  • Some suppliers can provide custom conjugation services

  • BSA-free versions of some MYO1D antibodies are available upon request

How do I analyze MYO1D expression in different cell populations or developmental stages?

To analyze MYO1D expression across different cell populations or developmental stages:

• Immunofluorescence co-staining:

  • Use MYO1D antibodies together with markers for specific cell types or developmental stages

  • For example, in oligodendrocyte studies, researchers used double immunostaining with O4, MBP, PLP (oligodendrocyte markers) and MYO1D to determine expression timing

  • Results showed that MYO1D expression coincides with PLP expression in cultured oligodendrocytes

• Quantitative analysis:

  • Calculate the percentage of MYO1D-positive cells within specific populations

  • In oligodendrocytes, the percentages of Myo1d-positive cells were:

    • 29.2% in O4+ cells

    • 62.1% in MBP+ cells

    • 100% in PLP+ cells

• Western blot analysis:

  • Compare MYO1D protein levels across different tissues or developmental timepoints

  • Normalize to appropriate housekeeping proteins

• RT-PCR for mRNA expression:

  • Quantify MYO1D mRNA levels relative to housekeeping genes (e.g., GAPDH)

  • siRNA treatment can reduce MYO1D expression by approximately 48% in cultured oligodendrocytes

• Proteomic analysis:

  • Mass spectrometry-based proteomic approaches can identify and quantify MYO1D

  • This approach revealed a 131% increase in MYO1D peptide counts in MYO1A knockout brush borders

What experimental approaches can distinguish between MYO1D and other closely related myosin family members?

Distinguishing between MYO1D and other myosin family members requires careful experimental design:

• Antibody selection:

  • Choose antibodies targeting unique epitopes specific to MYO1D

  • Validate antibody specificity against other myosins, especially closely related class I myosins

• Co-immunostaining:

  • Perform co-staining with antibodies against different myosins

  • For example, staining with anti-MYO1D and anti-MYO1A antibodies revealed distinct localization patterns in enterocytes

• Genetic approaches:

  • Use knockout/knockdown models specific to MYO1D

  • Analyze expression of other myosins in these models to identify potential compensatory mechanisms

  • Proteomic analysis of Myo1a KO brush borders showed increased MYO1D levels

• Domain-specific constructs:

  • Create domain-specific constructs to study the unique properties of MYO1D

  • MYO1D targeting requires both IQ and TH1 domains, whereas MYO1A requires only the TH1 domain

• Dynamic studies:

  • FRAP analysis can reveal differences in dynamics between myosin family members

  • MYO1D has different turnover kinetics compared to MYO1A, with MYO1A having a larger immobile fraction

• Table of key differences between MYO1D and MYO1A: