shtn1 Antibody

What is SHTN1 and why is it important in neuronal research?

SHTN1 (Shootin1) is an actin-binding protein that plays a pivotal role in cell development, morphogenesis, and other cellular functions. It contains a noncanonical WH2 domain and an upstream proline-rich region (PRR) that together are sufficient for actin interaction . SHTN1 is distinctively expressed in retinal ganglion cells (RGCs) during the period of active development and promotes neurite growth, complexity, and electrophysiological maturation . This protein is particularly significant in neuronal research because it represents a key molecular component in axonal development and neuronal connectivity formation.

What are the validated applications for SHTN1 antibodies?

Based on available research data, SHTN1 antibodies have been validated for several experimental applications:

• Western Blotting (WB): Successfully used with neuronal cell lines like Neuro-2a and rat brain tissue

• Immunofluorescence (IF): For cellular and tissue localization studies

• Immunohistochemistry (IHC): Validated on frozen and paraffin-embedded tissues

• ELISA: For quantitative protein detection

For optimal immunohistochemistry results with certain antibodies, antigen retrieval with TE buffer pH 9.0 is recommended .

How do I select the appropriate SHTN1 antibody for my experimental model?

When selecting a SHTN1 antibody, consider the following criteria:

• Species reactivity: Ensure compatibility with your experimental model. Available antibodies demonstrate reactivity with human, rat, mouse, horse, and monkey SHTN1

• Epitope specificity: Some antibodies target the N-terminal region (e.g., AA 21-130), which may be important depending on which domain you're studying

• Antibody type: Most available SHTN1 antibodies are polyclonal and raised in rabbit hosts

• Conjugation needs: Available options include unconjugated antibodies and various fluorophore conjugates (AbBy Fluor® 350, 488, 555, 594, 647, 680) depending on your detection method

For multi-label experiments, select conjugated antibodies compatible with your imaging setup and other markers.

How can I distinguish between SHTN1 isoforms in my experiments?

SHTN1 exists in different isoforms, including SHTN1L (long) and SHTN1S (short), with distinct functional properties. To distinguish between these isoforms:

• Use isoform-specific antibodies when available

• Employ high-resolution gel electrophoresis for Western blotting to separate the isoforms based on molecular weight

• Consider functional differences: SHTN1L shows stronger actin-binding activity than SHTN1S

• Evaluate subcellular localization patterns, as the isoforms show different distributions

• Validate findings with molecular approaches such as isoform-specific RT-PCR

A critical distinction between these isoforms is their interaction with actin: full-length SHTN1L directly interacts with G-actin in vitro, while SHTN1S shows substantially weaker interaction .

What is the optimal protocol for studying SHTN1's interaction with the actin cytoskeleton?

For robust analysis of SHTN1-actin interactions, implement the following protocol:

G-actin binding assay:

• Immunopurify EGFP-SHTN1 variants via GFP-Trap beads

• Wash in stringent buffer to eliminate endogenous actin interactions

• Re-equilibrate in G-buffer (5 mM Tris.HCl pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT)

• Clear G-actin by ultracentrifugation to remove oligomers

• Incubate ATP-actin monomers with immobilized SHTN1 variants

• Analyze binding through appropriate detection methods

F-actin cosedimentation assay:

• Incubate SHTN1 variants with pre-formed actin filaments

• Pellet by ultracentrifugation

• Analyze supernatant and pellet fractions by SDS-PAGE

• Calculate binding parameters (e.g., Kd values) through curve fitting

This combined approach allows comprehensive characterization of SHTN1's interactions with both monomeric and filamentous actin.

How does the autoinhibitory mechanism of SHTN1 affect experimental design?

SHTN1's actin-binding activity is intrinsically inhibited by its putative coiled-coil domain (CCD), with CCD-I functioning as the major autoinhibitory module . This autoinhibition mechanism has significant implications for experimental design:

• When studying SHTN1's actin-binding properties, consider generating CCD deletion mutants (particularly ΔCCD-I) to reveal the protein's full binding potential

• The affinity of CCD-I-deleted SHTN1 (1L-ΔCCD-I) for F-actin is substantially stronger than wild-type SHTN1L, with dissociation constants (Kd) of 21.86 nM versus 164.3 nM, respectively

• For subcellular localization studies, note that CCD deletions affect nuclear localization patterns of SHTN1

• When interpreting contradictory results about SHTN1 function, consider whether the experimental conditions might influence the autoinhibitory state

Understanding this regulatory mechanism is essential for accurate interpretation of SHTN1 functional data.

What methodological approaches can best elucidate SHTN1's role in neurite development?

To comprehensively investigate SHTN1's function in neurite development, consider these methodological approaches:

• Gain and loss of function studies:

  • shRNA-mediated knockdown of Shtn1

  • Overexpression of Shtn1 coding sequence (CDS)

  • Lentiviral delivery systems for genetic manipulation

• Morphological analysis:

  • Quantification of neurite length, branching complexity, and growth patterns

  • Time-lapse imaging to track dynamic changes in neurite development

• Functional assessment:

  • Patch clamp technique to measure electrophysiological properties and maturation

  • RNA sequencing to examine transcriptome changes associated with SHTN1 manipulation

• Model systems:

  • Direct somatic cell reprogramming to generate RGC-like neurons (iRGCs)

  • Primary neuronal cultures

  • In vivo developmental models

This multi-faceted approach provides comprehensive insights into how SHTN1 regulates neurite development at both molecular and cellular levels.

How can I investigate the nuclear localization of SHTN1 and its functional significance?

SHTN1 contains a conserved putative nuclear localization signal (NLS) between the PRR and WH2 domains that mediates its nuclear translocation . To study this aspect:

• Localization analysis:

  • Use immunofluorescence with validated antibodies to detect endogenous SHTN1

  • Create fusion constructs (e.g., EGFP-tagged domains) to study the localization of specific protein regions

  • Note that the EGFP-PRR-WH2 fragment containing the putative NLS localizes exclusively to the nucleus, while individual EGFP-PRR or EGFP-WH2 fragments distribute equally between nucleus and cytoplasm

• Regulatory mechanisms:

  • Investigate how CCDs regulate nuclear localization - deletion of CCD-I and CCD-II induces nuclear translocation of SHTN1

  • Create point mutations in the NLS sequence to confirm its functionality

• Functional studies:

  • Compare cytoplasmic versus nuclear functions of SHTN1

  • Investigate potential interactions with nuclear proteins or chromatin

Understanding the nuclear-cytoplasmic distribution of SHTN1 may reveal previously unexplored functions beyond its established role in actin cytoskeletal regulation.

What are common pitfalls when using SHTN1 antibodies and how can they be addressed?

When working with SHTN1 antibodies, researchers may encounter several challenges:

• Background signal in immunostaining:

  • Optimize blocking conditions (try different blocking agents: BSA, serum, commercial blockers)

  • Increase washing duration and frequency

  • For human tissues with high background, consider antigen retrieval with TE buffer pH 9.0

• Multiple bands in Western blot:

  • Determine if bands represent isoforms (SHTN1L vs SHTN1S) or degradation products

  • Use freshly prepared samples with protease inhibitors

  • Include positive controls (e.g., Neuro-2a cells, rat brain tissue)

• Inconsistent immunoprecipitation results:

  • When studying actin interactions, use stringent washing conditions followed by re-equilibration in appropriate buffer (e.g., G-buffer for G-actin studies)

  • Consider the autoinhibitory effect of CCD-I when analyzing binding results

• Low signal in neuronal cultures:

  • Verify expression timing in your model system, as SHTN1 expression may be developmentally regulated

  • Optimize fixation protocols to preserve epitope accessibility

How can I quantitatively assess SHTN1 expression or localization changes?

For rigorous quantitative analysis of SHTN1:

• Western blot quantification:

  • Use appropriate loading controls

  • Employ standard curves with recombinant protein when absolute quantification is needed

  • Analyze multiple biological replicates

• Immunofluorescence quantification:

  • Maintain consistent imaging parameters across all samples

  • Measure integrated intensity rather than peak intensity

  • Normalize to appropriate cell markers

  • Perform z-stack imaging for accurate 3D quantification

• Subcellular distribution analysis:

  • Calculate nuclear/cytoplasmic ratios based on signal intensity

  • Consider that CCD-I deletion promotes nuclear localization of SHTN1S but has less effect on SHTN1L

  • Use computational image analysis for unbiased assessment

• Statistical considerations:

  • Analyze sufficient numbers of cells/samples for statistical power

  • Apply appropriate statistical tests based on data distribution

  • Consider blind analysis to prevent bias

What emerging techniques could advance our understanding of SHTN1 function?

Several cutting-edge approaches show promise for SHTN1 research:

• CRISPR-Cas9 genome editing:

  • Generate endogenous tagged SHTN1 for live imaging without overexpression artifacts

  • Create domain-specific mutations to dissect functional contributions

• Super-resolution microscopy:

  • Visualize SHTN1-actin interactions at nanoscale resolution

  • Track dynamic changes during neurite development

• Proximity labeling approaches:

  • Identify the complete interactome of SHTN1 in different cellular compartments

  • Discover novel binding partners beyond the actin cytoskeleton

• Single-cell transcriptomics:

  • Characterize cell-specific expression patterns of SHTN1 isoforms

  • Identify co-regulated gene networks

These advanced techniques could reveal new aspects of SHTN1 function beyond its established role in neurite development and actin binding.

How might SHTN1 research contribute to understanding neurological disorders?

Given SHTN1's critical role in neurite development and axonal guidance , its dysfunction could potentially contribute to various neurological conditions:

• Neurodevelopmental disorders:

  • Investigation of SHTN1 expression and function in autism spectrum disorders, intellectual disability, or schizophrenia

  • Analysis of SHTN1 genetic variants in patient cohorts

• Neurodegeneration:

  • Examination of SHTN1's potential role in axonal maintenance and regeneration

  • Therapeutic targeting to promote neural repair

• Visual system disorders:

  • Given SHTN1's role in retinal ganglion cell development , investigation of its contribution to optic neuropathies

  • Potential application in regenerative approaches for vision loss

Further research into these areas could identify SHTN1 as a therapeutic target or biomarker for neurological conditions with disrupted neuronal connectivity.