spire2 Antibody

What is SPIRE2 and why is it important in cellular research?

SPIRE2 (Spire Homolog 2) functions as an actin nucleation factor that remains associated with the slow-growing pointed end of newly formed actin filaments . It plays critical roles in several cellular processes:

• Mediates intracellular vesicle transport along actin fibers, establishing a novel link between actin cytoskeleton dynamics and intracellular transport mechanisms

• Required for asymmetric spindle positioning and asymmetric cell division during meiosis

• Essential for normal formation of the cleavage furrow and polar body extrusion during female germ cell meiosis

• Acts in the nucleus to promote assembly of nuclear actin filaments in response to DNA damage, facilitating movement of chromatin and repair factors after DNA damage

Understanding SPIRE2's functions provides insights into fundamental cellular processes related to cytoskeletal organization, vesicular trafficking, and cell division.

What types of SPIRE2 antibodies are available for research applications?

Research-grade SPIRE2 antibodies are available in several formats to accommodate diverse experimental needs:

Most commonly, researchers utilize rabbit polyclonal antibodies directed against different epitopes of human SPIRE2, with immunogens typically derived from amino acid sequences between positions 79-388 , 285-313 , or 355-563 .

What are the validated applications for SPIRE2 antibodies?

SPIRE2 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Successfully detects SPIRE2 at approximately 79-80 kDa in various cell and tissue extracts

• Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative detection of SPIRE2 with high sensitivity

• Immunohistochemistry (IHC): Enables visualization of SPIRE2 in tissue sections

• Immunofluorescence (IF): Allows subcellular localization studies of SPIRE2

• Dot Blot: Offers a rapid screening method for SPIRE2 detection

Recommended dilutions vary by application and specific antibody, typically ranging from 1:500-1:2000 for Western blotting and up to 1:80000 for ELISA .

What are the recommended storage conditions for SPIRE2 antibodies?

To maintain antibody integrity and activity, follow these storage guidelines:

• Store at -20°C for short-term preservation or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Many SPIRE2 antibodies are supplied in stabilizing buffers containing glycerol (typically 50%) and preservatives like sodium azide (0.02-0.03%)

• Some antibodies are provided in lyophilized form and require reconstitution in sterile distilled water with 50% glycerol before use

• Reconstituted antibodies should be aliquoted to minimize freeze-thaw cycles

Following proper storage protocols ensures optimal antibody performance and extends shelf life.

How should researchers optimize Western blot protocols for SPIRE2 detection?

Western blot optimization for SPIRE2 requires attention to several technical parameters:

• Sample preparation: SPIRE2 has been successfully detected in various cellular extracts including U-87MG, A431, HeLa cells, and mouse/rat tissues (cerebellum, liver, brain, testis)

• Dilution optimization:

  • Start with manufacturer's recommended range (typically 1:500-1:2000)

  • Perform titration experiments if signal strength is suboptimal

  • For Proteintech's antibody (#17757-1-AP), optimal results were achieved at 1:500 dilution

• Protocol parameters:

  • Incubation: Room temperature for 1.5 hours shows good results with some antibodies

  • Expected molecular weight: Look for bands at approximately 79-80 kDa

  • Positive controls: Mouse cerebellum tissue has been validated as an effective positive control

• Buffer composition: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) is commonly used as a storage buffer for antibodies before dilution in blocking solution

Validation data shows that mouse cerebellum tissue subjected to SDS-PAGE followed by Western blot with antibody diluted at 1:500 and incubated at room temperature for 1.5 hours produces clear detection of SPIRE2 .

What considerations are important when selecting tissue samples for SPIRE2 studies?

When selecting appropriate tissues for SPIRE2 research, consider:

• Validated tissue types: Several tissues have been confirmed to express SPIRE2 at detectable levels:

  • Neural tissues: cerebellum, brain (mouse and rat)

  • Reproductive tissues: testis (mouse and rat)

  • Other tissues: liver (mouse)

• Subcellular localization: SPIRE2 can be found in multiple cellular compartments:

  • Cell membrane

  • Cytoplasm (cytoplasmic side)

  • Cytoplasmic vesicle membrane

  • Peripheral membrane protein regions

  • Cytoskeleton

  • Cytosol

  • Perinuclear region

• Sample processing: Fresh or properly preserved tissues yield best results; avoid repeated freezing and thawing of samples

• Control selection: Include both positive controls (tissues known to express SPIRE2) and negative controls to validate antibody specificity

Understanding the expected distribution pattern of SPIRE2 helps in experimental design and interpretation of results.

How can researchers validate the specificity of their SPIRE2 antibody?

Antibody validation is critical for ensuring reliable results. For SPIRE2 antibodies, consider these validation approaches:

• Western blot analysis with known positive controls:

  • Use established positive controls such as U-87MG, A431, HeLa cells, mouse cerebellum, mouse brain, mouse testis, rat brain, or rat testis

  • Confirm detection at the expected molecular weight (approximately 79-80 kDa)

• Immunogen competition assays:

  • Pre-incubate antibody with the immunizing peptide

  • Compare results with and without peptide competition

  • Signal should be reduced or eliminated in the presence of the specific immunizing peptide

• Multiple antibody comparison:

  • Compare results using antibodies raised against different epitopes of SPIRE2

  • Consistent results across antibodies targeting different regions (e.g., AA 79-388, 285-313, 355-563) increase confidence in specificity

• Cross-reactivity testing:

  • Test antibodies against recombinant SPIRE1 (the closest paralog)

  • Verify minimal cross-reactivity with related proteins

• Citations and literature validation:

  • Review published studies using the same antibody

  • Some SPIRE2 antibodies have been cited in publications validating their use in Western blot, immunoprecipitation, immunofluorescence, and immunohistochemistry

What are the methodological considerations for studying SPIRE2's role in actin nucleation?

When investigating SPIRE2's function in actin nucleation, consider these methodological approaches:

• Co-localization studies:

  • Use immunofluorescence with SPIRE2 antibodies together with actin markers

  • Examine co-localization at the slow-growing pointed end of actin filaments

  • FITC-conjugated SPIRE2 antibodies may be particularly useful for direct visualization

• Functional assays:

  • Combine antibody detection with actin polymerization assays

  • Correlate SPIRE2 localization with newly formed actin filaments

  • Consider the finding that SPIRE2 "remains associated with the slow-growing pointed end of the new filament"

• Nuclear actin filament assembly:

  • Investigate SPIRE2's role in promoting assembly of nuclear actin filaments in response to DNA damage

  • Examine how SPIRE2 facilitates movement of chromatin and repair factors after DNA damage

• Complementary techniques:

  • Combine antibody-based detection with siRNA knockdown or overexpression systems

  • Use live cell imaging with tagged proteins in conjunction with fixed-cell antibody staining

• Context-specific considerations:

  • Study SPIRE2 in relation to other actin nucleation factors

  • Investigate the role of SPIRE2 in different cell types, as function may vary between tissues

How should researchers approach SPIRE2 studies in reproductive biology contexts?

Given SPIRE2's critical role in meiosis and reproduction, special considerations apply to studies in this context:

• Meiotic spindle positioning:

  • SPIRE2 is required for asymmetric spindle positioning and asymmetric cell division during meiosis

  • Use immunofluorescence to track SPIRE2 localization during different stages of meiosis

• Cleavage furrow formation:

  • SPIRE2 is essential for normal formation of the cleavage furrow

  • Study SPIRE2 dynamics during cytokinesis using time-lapse microscopy in conjunction with antibody staining

• Polar body extrusion:

  • SPIRE2 is required for polar body extrusion during female germ cell meiosis

  • Use antibodies to track SPIRE2 localization during oocyte maturation

• Tissue selection:

  • Mouse and rat testis have been validated as positive controls for SPIRE2 expression

  • Consider using these tissues as reference standards when studying SPIRE2 in reproductive contexts

• Controls and validation:

  • Include age-matched controls when studying reproductive tissues

  • Consider hormonal status and reproductive cycle when designing experiments

Using these methodological approaches will help researchers accurately characterize SPIRE2's role in reproductive biology.

How can researchers address weak or absent signals in SPIRE2 Western blots?

When facing detection challenges with SPIRE2 antibodies in Western blotting, consider these troubleshooting approaches:

• Sample preparation optimization:

  • Ensure complete protein extraction using appropriate lysis buffers

  • Add protease inhibitors to prevent SPIRE2 degradation

  • Freshly prepare samples when possible

• Antibody dilution adjustment:

  • If using the recommended 1:500-1:2000 dilution range yields weak signals, try increasing antibody concentration

  • For Proteintech's antibody (#17757-1-AP), validated results were achieved at 1:500 dilution

• Incubation conditions:

  • Extend primary antibody incubation time (overnight at 4°C instead of 1.5 hours at room temperature)

  • Optimize secondary antibody concentration and incubation period

• Signal enhancement strategies:

  • Use more sensitive detection systems (e.g., enhanced chemiluminescence)

  • Consider amplification systems for low-abundance targets

  • Try reducing washing stringency slightly while maintaining specificity

• Positive control inclusion:

  • Always run validated positive controls like mouse cerebellum tissue

  • If positive control works but sample doesn't, this indicates a sample-specific issue rather than antibody failure

If signal remains problematic, consider switching to a different SPIRE2 antibody targeting an alternative epitope.

What are the key considerations for multiplexing SPIRE2 antibodies with other markers?

Multiplexing SPIRE2 detection with other cellular markers requires careful planning:

• Antibody compatibility:

  • Choose primary antibodies from different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider directly conjugated versions or sequential staining protocols

• Fluorophore selection for immunofluorescence:

  • When using FITC-conjugated SPIRE2 antibodies , choose complementary fluorophores with minimal spectral overlap

  • Account for SPIRE2's multiple subcellular localizations (cell membrane, cytoplasm, cytoskeletal associations, perinuclear region)

• Co-staining recommendations:

  • Consider co-staining with actin markers to investigate SPIRE2's role in actin nucleation

  • For vesicular transport studies, combine with vesicle markers

  • For meiosis research, pair with spindle and chromosomal markers

• Sequential detection protocols:

  • When primary antibodies are from the same species, use sequential staining with intermediate blocking steps

  • Consider spectral unmixing techniques for complex multiplexing

• Controls for multiplexing:

  • Include single-stained controls to assess bleed-through

  • Use isotype controls to verify specificity of each antibody in the multiplex panel

Careful optimization of multiplex protocols will yield the most informative results when studying SPIRE2 in complex cellular contexts.

How can SPIRE2 antibodies be utilized to study intracellular vesicle transport?

SPIRE2's role in intracellular vesicle transport can be investigated using several antibody-based approaches:

• Co-localization with vesicle markers:

  • Use SPIRE2 antibodies in combination with markers for different vesicle populations

  • SPIRE2 is "involved in intracellular vesicle transport along actin fibers, providing a novel link between actin cytoskeleton dynamics and intracellular transport"

• Live-cell trafficking studies:

  • Combine fixed-cell antibody staining with live-cell imaging of vesicle movements

  • Correlate SPIRE2 distribution with vesicle dynamics

• Subcellular fractionation validation:

  • Use Western blotting with SPIRE2 antibodies on subcellular fractions

  • Confirm SPIRE2 enrichment in vesicular fractions

  • SPIRE2 is found in "cytoplasmic vesicle membrane" compartments

• Perturbation approaches:

  • Study changes in SPIRE2 localization after cytoskeletal disruption

  • Examine how vesicle transport is affected when SPIRE2 is depleted or overexpressed

• High-resolution microscopy:

  • Apply super-resolution techniques with SPIRE2 antibodies to visualize association with vesicle transport machinery

  • Study the interface between vesicles, SPIRE2, and actin filaments

These methodologies will help elucidate SPIRE2's precise role in coordinating vesicle movement along actin filaments.

What methodological approaches are recommended for studying SPIRE2's nuclear functions?

SPIRE2's nuclear role in actin filament assembly after DNA damage requires specialized experimental approaches:

• Nuclear-cytoplasmic fractionation:

  • Use Western blotting with SPIRE2 antibodies on nuclear and cytoplasmic fractions

  • Validate the presence of SPIRE2 in nuclear compartments

  • Compare distribution before and after DNA damage induction

• DNA damage response studies:

  • Induce DNA damage using established methods (UV, radiomimetic drugs, etc.)

  • Track SPIRE2 localization using immunofluorescence before and after damage

  • SPIRE2 "promotes assembly of nuclear actin filaments in response to DNA damage in order to facilitate movement of chromatin and repair factors after DNA damage"

• Co-localization with DNA repair factors:

  • Perform dual immunofluorescence with SPIRE2 antibodies and markers of DNA damage/repair

  • Examine temporal dynamics of these associations

• Nuclear actin visualization:

  • Combine SPIRE2 antibody staining with specialized probes for nuclear actin

  • Study how SPIRE2 affects nuclear actin polymerization after DNA damage

• Functional readouts:

  • Correlate SPIRE2 activity with measurements of DNA repair efficiency

  • Assess how manipulation of SPIRE2 affects chromatin mobility after damage

These approaches will help characterize SPIRE2's role in nuclear processes, particularly in the context of DNA damage response.

How should researchers design experiments to study SPIRE2 in meiotic processes?

Given SPIRE2's critical functions in meiosis , specialized experimental designs are necessary:

• Developmental timing considerations:

  • Study SPIRE2 at specific stages of meiosis when asymmetric spindle positioning occurs

  • Track temporal dynamics during cleavage furrow formation and polar body extrusion

• Imaging approaches:

  • Use high-resolution imaging of fixed samples with SPIRE2 antibodies

  • Combine with markers for meiotic spindles, chromosomes, and cortical cytoskeleton

  • Consider z-stack acquisitions to capture 3D organization during polar body formation

• Functional perturbation studies:

  • Complement antibody visualization with knockdown/knockout approaches

  • Assess consequences for asymmetric spindle positioning, cleavage furrow formation, and polar body extrusion

• Sample selection considerations:

  • Choose appropriate models (mouse oocytes are well-established)

  • Use proper collection and fixation techniques that preserve meiotic structures

• Quantitative analysis methods:

  • Develop metrics for spindle positioning, cleavage furrow progression, and polar body size

  • Compare wild-type and SPIRE2-perturbed samples quantitatively

These methodological considerations will facilitate rigorous investigation of SPIRE2's roles in meiotic processes.

What emerging technologies might enhance SPIRE2 antibody applications?

Several technological advances could expand the utility of SPIRE2 antibodies in research:

• Super-resolution microscopy integration:

  • Apply techniques like STORM, PALM, or STED with SPIRE2 antibodies

  • Resolve nanoscale organization of SPIRE2 at actin filament pointed ends

  • Visualize SPIRE2-mediated interactions between vesicles and cytoskeleton

• Proximity labeling approaches:

  • Combine SPIRE2 antibodies with proximity labeling techniques

  • Identify novel interaction partners in different cellular contexts

  • Map the spatial relationships between SPIRE2 and related proteins

• Single-cell analysis methods:

  • Apply SPIRE2 antibodies in single-cell proteomic workflows

  • Correlate SPIRE2 levels with cellular states and functions

  • Identify cell-to-cell variations in SPIRE2 expression and localization

• Tissue clearing and 3D imaging:

  • Use SPIRE2 antibodies with tissue clearing techniques

  • Study SPIRE2 distribution in intact tissues and organs

  • Examine SPIRE2's role in complex multicellular contexts

• CRISPR-based tagging combined with antibody validation:

  • Use genome editing to tag endogenous SPIRE2

  • Validate antibody specificity against tagged proteins

  • Develop improved reagents for SPIRE2 research

These technological approaches will expand our understanding of SPIRE2's diverse cellular functions.

How can researchers address the methodological challenges in studying SPIRE2's multi-functional nature?

SPIRE2's involvement in multiple cellular processes presents unique experimental challenges:

• Context-specific function analysis:

  • Design experiments that distinguish between SPIRE2's roles in different cellular processes

  • Use cell type-specific approaches to isolate particular functions

  • Compare SPIRE2 behavior across different biological contexts

• Temporal regulation studies:

  • Implement time-resolved approaches to track SPIRE2 dynamics

  • Determine how SPIRE2 switches between different functional modes

  • Correlate SPIRE2 activity with cell cycle or developmental stages

• Multimodal data integration:

  • Combine antibody-based imaging with functional assays

  • Integrate proteomic, genomic, and imaging datasets

  • Develop computational models of SPIRE2's diverse activities

• Domain-specific antibody application:

  • Utilize antibodies targeting different domains of SPIRE2

  • Map domain-specific functions in various cellular contexts

  • Distinguish between the activities of different SPIRE2 structural elements

• Comparative analysis with SPIRE1:

  • Study SPIRE2 alongside its paralog SPIRE1

  • Determine unique and overlapping functions

  • Investigate how these proteins collaborate in processes like nuclear actin assembly

These methodological strategies will help untangle SPIRE2's complex and multifaceted cellular roles.