smcr8a Antibody

What is SMCR8 and why is it important in cellular research?

SMCR8 is a protein originally identified in patients with Smith-Magenis syndrome, a rare developmental disorder . It has gained significant research interest due to its role in multiple cellular processes. SMCR8 forms a complex with C9orf72 and WDR41, functioning as a guanine nucleotide exchange factor (GEF) for Rab8a and Rab39b, which are critical for autophagosome maturation .

The importance of SMCR8 in research is underscored by its association with:

• Autophagy regulation through the ULK1 complex

• Lysosomal signaling pathways

• Neurodegenerative diseases through its association with C9orf72 (linked to amyotrophic lateral sclerosis and frontotemporal dementia)

• mTORC1 signaling as it promotes phosphorylation of mTORC1 substrates

Research indicates SMCR8 has dual localization in both cytoplasm and nucleus, where it associates with chromatin and negatively regulates expression of ULK1 and WIPI2 genes .

What are the key applications for SMCR8 antibodies in research?

Based on current literature and product information, SMCR8 antibodies are primarily utilized in the following research applications:

SMCR8 antibodies are particularly valuable for investigating autophagy pathways, neurodegenerative disease mechanisms, and the regulation of mTORC1 signaling .

How should researchers validate SMCR8 antibodies before use in critical experiments?

Thorough validation of SMCR8 antibodies is essential for reliable research outcomes. A comprehensive validation should include:

• Species-specific validation: Confirm reactivity in your experimental model (human, mouse, rat) as SMCR8 sequence conservation may vary .

• Application-specific testing: Each application (WB, IP, ICC) requires separate validation as antibodies that work well in one application may fail in another .

• Positive controls: Use cell lines known to express SMCR8 (e.g., HeLa, HEK-293T) as demonstrated in validation studies .

• Negative controls:

  • Employ knockdown/knockout models where SMCR8 is depleted

  • Use isotype controls to assess non-specific binding

  • Test in tissues/cells known not to express SMCR8

• Molecular weight verification: Confirm detection at the expected molecular weight (140-150 kDa) .

• Fixation/permeabilization assessment: If using for intracellular staining, test performance with different fixation protocols as some antibodies fail after certain fixation methods .

• Epitope mapping: Understand which region of SMCR8 the antibody recognizes (e.g., some target areas within aa 600-650) .

A methodical approach to validation saves time and resources while ensuring experimental reliability.

What are the optimal conditions for using SMCR8 antibodies in western blotting?

Optimizing western blotting conditions for SMCR8 detection requires attention to several key parameters:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylated SMCR8 forms

  • Sonicate briefly to shear DNA and reduce sample viscosity

• Gel selection and running conditions:

  • Use 6-8% gels or gradient gels (4-12%) to properly resolve the high molecular weight SMCR8 (140-150 kDa)

  • Run gel at lower voltage (80-100V) for better resolution of large proteins

• Transfer conditions:

  • Use wet transfer system for large proteins

  • Extend transfer time (2-3 hours) or perform overnight at low voltage/amperage

  • Consider adding SDS (0.02%) to transfer buffer to facilitate large protein transfer

• Blocking and antibody incubation:

  • Recommended dilution: 1:1000

  • Incubate primary antibody overnight at 4°C for optimal sensitivity

  • Use 5% BSA in TBST for blocking and antibody dilution to reduce background

• Detection:

  • Enhanced chemiluminescence (ECL) with extended exposure times (3 minutes shown to be effective)

  • Consider signal enhancement systems for low-abundance detection

• Controls to include:

  • Molecular weight marker

  • Positive control lysate (HeLa or HEK-293T cells)

  • Loading control (preferably not in the same molecular weight range as SMCR8)

Following these guidelines will help ensure specific detection of SMCR8 protein in western blot experiments.

How can researchers effectively use SMCR8 antibodies to study its interactions with the autophagy machinery?

Investigating SMCR8's role in autophagy requires sophisticated experimental approaches:

• Co-immunoprecipitation (Co-IP) studies:

  • Use SMCR8 antibodies (1:50 dilution) to pull down protein complexes

  • Probe for known interactors: C9orf72, WDR41, ULK1, FIP200, ATG13

  • Include crosslinking steps (e.g., DSP) to capture transient interactions

  • Use reciprocal IP (pull down with ULK1 antibody, probe for SMCR8) to confirm interactions

• Proximity ligation assay (PLA):

  • Visualize endogenous protein-protein interactions in situ

  • Combine SMCR8 antibody with antibodies against interacting partners

  • Quantify interaction signals under different autophagy states (basal, starvation, inhibition)

• Phosphorylation status analysis:

  • Use phospho-specific antibodies or general phospho-detection after SMCR8 immunoprecipitation

  • Monitor changes in phosphorylation after treatment with TBK1/ULK1 activators/inhibitors

  • Perform lambda phosphatase treatment to confirm specificity of phosphorylation signals

• Fractionation experiments:

  • Separate cytoplasmic, nuclear, and autophagosome-enriched fractions

  • Use SMCR8 antibody to track localization changes during autophagy induction

  • Include markers for each fraction (e.g., GAPDH, Histone H3, LC3-II)

• Live-cell imaging with tagged SMCR8:

  • Validate antibody specificity against tagged constructs

  • Compare endogenous staining with tagged protein localization

These methodologies can provide comprehensive insights into how SMCR8 regulates autophagy through its interactions with key machinery components .

What experimental approaches can be used to study the dual function of SMCR8 in cytoplasmic and nuclear compartments?

SMCR8's dual localization presents unique research challenges requiring specialized techniques:

• Subcellular fractionation and western blotting:

  • Perform differential centrifugation to isolate nuclear, cytoplasmic, and chromatin-bound fractions

  • Use SMCR8 antibody to detect protein distribution across fractions

  • Include compartment-specific markers (nuclear: Lamin B1; cytoplasmic: GAPDH; chromatin: Histone H3)

  • Quantify relative distribution between compartments under different conditions

• Chromatin immunoprecipitation (ChIP):

  • Use SMCR8 antibody to precipitate chromatin fragments

  • Perform qPCR for ULK1 and WIPI2 gene promoters to validate transcriptional regulation

  • Sequence precipitated DNA (ChIP-seq) to identify genome-wide binding sites

• Immunofluorescence microscopy with co-localization analysis:

  • Perform dual staining with SMCR8 antibody and compartment markers

  • Use super-resolution microscopy for detailed localization

  • Quantify co-localization coefficients (Pearson's or Mander's)

  • Compare localization patterns under different conditions (nutrient status, cell cycle stage)

• Nuclear-cytoplasmic shuttling experiments:

  • Treat cells with nuclear export inhibitors (Leptomycin B)

  • Monitor SMCR8 redistribution using the antibody

  • Identify potential nuclear localization and export signals

• Protein domain analysis:

  • Create domain-specific deletions and use antibodies to validate expression

  • Determine which domains influence subcellular localization

  • Correlate localization with function in each compartment

These approaches can elucidate how SMCR8 exerts its distinct functions in regulating autophagy in the cytoplasm while controlling gene expression in the nucleus .

How should researchers address potential cross-reactivity issues when using SMCR8 antibodies?

Cross-reactivity is a significant concern in antibody-based research. For SMCR8 antibodies, implement these rigorous approaches:

• Epitope analysis and validation:

  • Determine the exact epitope recognized by your antibody (e.g., within aa 600-650)

  • Conduct BLAST analysis of the epitope sequence to identify potential cross-reactive proteins

  • Consider testing multiple antibodies targeting different SMCR8 epitopes

• Knockout/knockdown validation:

  • Create SMCR8 knockout or knockdown models using CRISPR-Cas9 or siRNA

  • Any remaining signal after complete knockout indicates cross-reactivity

  • Include these controls in publications to demonstrate antibody specificity

• Preabsorption controls:

  • Preincubate antibody with purified antigen or immunizing peptide

  • This should eliminate specific signal while leaving cross-reactive signals intact

  • Compare with non-preabsorbed antibody to identify non-specific bands

• Species-specific considerations:

  • Verify antibody performance in each species (human, mouse, rat)

  • Be aware that antibodies raised against human SMCR8 may perform differently in rodent models

• Advanced verification techniques:

  • Use orthogonal detection methods (mass spectrometry after IP)

  • Employ multiple antibodies targeting different epitopes

  • Perform immunodepletion experiments to confirm specificity

• Signal quantification and reporting:

  • Document all bands observed, not just the expected 140-150 kDa band

  • Report potential cross-reactive signals in publications

  • Use appropriate statistical analyses for quantitative comparisons

Implementing these approaches ensures reliable interpretation of experimental results and enhances reproducibility across research studies .

What strategies can researchers employ to resolve contradictory data when studying SMCR8 function with antibodies?

When faced with contradictory data regarding SMCR8 function, consider these methodological approaches:

• Antibody validation reassessment:

  • Thoroughly revalidate all antibodies used in the contradictory studies

  • Determine if different antibodies recognize distinct epitopes or isoforms

  • Conduct parallel experiments with multiple validated antibodies

• Post-translational modification analysis:

  • SMCR8 is subject to phosphorylation by TBK1 and ULK1

  • Different antibodies may have varying sensitivities to phosphorylated forms

  • Use phosphatase treatment to determine if contradictions arise from detection of different modification states

• Context-dependent function investigation:

  • SMCR8 functions differently in different cellular compartments

  • Systematically test conditions (cell types, nutrient status, stress conditions)

  • Map contradictory findings to specific cellular contexts

• Interactome analysis:

  • Perform IP-mass spectrometry to identify all SMCR8 binding partners

  • Different protein-protein interactions may explain context-dependent functions

  • Compare interactomes under conditions that produce contradictory results

• Isoform-specific analysis:

  • Determine if different splice variants exist with distinct functions

  • Design isoform-specific detection strategies

  • Express individual isoforms in knockout backgrounds to test function

• Temporal dynamics consideration:

  • SMCR8 may have time-dependent functions in autophagy

  • Perform detailed time-course experiments after autophagy induction

  • Use live-cell imaging with temporal resolution to track dynamics

• Data integration and statistical analysis:

  • Create comprehensive models incorporating all experimental variables

  • Apply appropriate statistical methods to identify significant factors explaining variability

  • Use Bayesian approaches to update hypotheses based on accumulated evidence

This systematic approach can reconcile apparently contradictory findings and lead to a more nuanced understanding of SMCR8's multifaceted roles .

How can researchers optimize SMCR8 antibody-based assays for studying its role in neurodegenerative disease models?

Optimizing SMCR8 antibody applications for neurodegenerative disease research requires specialized approaches:

• Model system selection and validation:

  • Test antibody performance in relevant models (iPSC-derived neurons, organoids, animal models)

  • Validate detection in post-mortem human brain tissue with appropriate controls

  • Optimize fixation protocols for neural tissues (often requiring longer fixation times)

• Co-detection with disease markers:

  • Establish multiplex protocols combining SMCR8 antibody with:

    • C9orf72 (ALS/FTD-associated protein)

    • TDP-43 (pathological inclusions)

    • p62/SQSTM1 (autophagy substrate that accumulates in disease)

  • Optimize antibody combinations to avoid cross-reactivity

• Quantitative analysis adaptations:

  • Develop image analysis pipelines for co-localization with disease markers

  • Use high-content imaging for large-scale phenotypic analysis

  • Implement machine learning approaches for pattern recognition in complex tissues

• Age-dependent and region-specific analyses:

  • Compare SMCR8 levels/localization across brain regions

  • Conduct age-dependent studies in models of neurodegenerative diseases

  • Use laser capture microdissection combined with western blotting for region-specific analysis

• Patient-derived sample considerations:

  • Optimize protocols for limited and precious patient material

  • Develop more sensitive detection methods (e.g., proximity extension assays)

  • Create standardized protocols that control for post-mortem interval effects

• Functional readouts in disease models:

  • Correlate SMCR8 antibody staining patterns with:

    • Autophagy dysfunction markers

    • Lysosomal function assays

    • Neuronal health indicators

• Data presentation and analysis:

  • Use quantitative image analysis to present unbiased data

  • Include comprehensive controls in each experiment

  • Apply statistical methods appropriate for highly variable biological samples

These optimizations can help researchers elucidate SMCR8's role in neurodegenerative diseases through its connections with C9orf72 and autophagy regulation .

How should researchers interpret changes in SMCR8 protein levels in relation to autophagy dysfunction?

Interpreting SMCR8 protein level changes requires careful consideration of its complex roles in autophagy:

• Baseline establishment:

  • Determine normal SMCR8 expression levels across relevant cell types using validated antibodies

  • Create quantitative western blot standards using recombinant SMCR8

  • Document cell type-specific variations to establish reference ranges

• Contextual analysis framework:

  • SMCR8 operates as both a positive and negative regulator of autophagy depending on context

  • In the C9orf72-SMCR8-WDR41 complex: promotes autophagosome maturation

  • When interacting with ULK1: inhibits autophagy initiation

  • Map observed changes to specific regulatory mechanisms

• Multi-parameter analysis:

  • Always assess SMCR8 levels alongside:

    • Autophagy flux markers (LC3-II/I ratio, p62 levels)

    • Partner proteins (C9orf72, WDR41)

    • Upstream regulators (mTOR, AMPK activity)

    • Downstream effects (lysosomal function)

• Phosphorylation status integration:

  • Interpret SMCR8 data in context of its phosphorylation state

  • Increased phosphorylation by TBK1/ULK1 may indicate altered function rather than expression

  • Use phospho-specific antibodies or phosphorylation-dependent mobility shifts

• Subcellular distribution changes:

  • Analyze nuclear vs. cytoplasmic distribution changes

  • Shifts in localization may indicate altered function without total protein changes

  • Use cell fractionation and imaging approaches to quantify redistribution

• Temporal dynamics consideration:

  • Changes in SMCR8 levels may follow distinct kinetics during autophagy induction

  • Establish time-course experiments to capture dynamic changes

  • Use pulse-chase methods to determine if protein stability is altered

Understanding these complex relationships allows researchers to more accurately interpret changes in SMCR8 protein levels and their implications for autophagy dysfunction .

What are the critical considerations when designing experiments to study SMCR8 phosphorylation status using antibodies?

Studying SMCR8 phosphorylation requires specialized experimental design:

• Phosphorylation site mapping and antibody selection:

  • SMCR8 is phosphorylated by TBK1 and ULK1 at multiple sites

  • Use phospho-specific antibodies when available

  • For sites lacking specific antibodies, consider:

    • Phospho-motif antibodies (e.g., phospho-Ser/Thr)

    • Custom phospho-specific antibody development

    • Mass spectrometry-based phosphopeptide detection

• Sample preparation optimization:

  • Include phosphatase inhibitors in all lysis buffers (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Maintain samples at 4°C throughout processing

  • Consider parallel samples with/without lambda phosphatase treatment as controls

  • Use Phos-tag™ gels to enhance mobility shifts of phosphorylated proteins

• Experimental design for kinase-substrate relationships:

  • Manipulate TBK1 and ULK1 activity through:

    • Small molecule inhibitors (MRT67307 for TBK1, SBI-0206965 for ULK1)

    • Genetic approaches (kinase-dead mutants, CRISPR knockout)

    • Physiological activation (starvation, LPS treatment)

  • Monitor SMCR8 phosphorylation status in response to these manipulations

• Functional correlation analysis:

  • Create phosphomimetic (S→D/E) and phospho-deficient (S→A) SMCR8 mutants

  • Compare their localization and function to wild-type SMCR8

  • Use antibodies to monitor endogenous SMCR8 alongside these constructs

• Technical considerations for detection:

  • Phosphorylation may alter antibody epitope accessibility

  • Test antibody performance with both phosphorylated and dephosphorylated samples

  • Consider 2D gel electrophoresis to separate phospho-isoforms

  • Use multiple antibodies recognizing different SMCR8 regions

• Data quantification and statistical analysis:

  • Quantify phosphorylation signal relative to total SMCR8

  • Apply appropriate statistical tests for comparing phosphorylation levels

  • Consider stoichiometry (what percentage of SMCR8 is phosphorylated) in interpretations

These methodological considerations enhance the reliability of phosphorylation studies and allow meaningful interpretation of SMCR8 regulation .