sigmar1 Antibody

How should researchers interpret differences in SIGMAR1 staining patterns across different studies?

Variations in SIGMAR1 staining patterns often stem from three factors: (1) different antibodies recognizing distinct epitopes, (2) cell type-specific expression patterns, and (3) methodological differences in sample preparation. For example, some studies employed special treatments (e.g., 0.02% SDS for 10 minutes) to visualize SIGMAR1 in detergent-insoluble lipid microdomains . When comparing results across studies, researchers should carefully consider the exact antibody used, its validation method, and specific sample preparation techniques. The comprehensive table compiled by researchers shows at least 17 different experimental approaches for detecting SIGMAR1 in various subcellular compartments .

What controls should be included when using SIGMAR1 antibodies for the first time?

For proper validation of SIGMAR1 antibody specificity, include:

• Positive controls: Cell lines known to express SIGMAR1 (HEK293T, NSC34, Neuro2a, or SH-SY5Y cells)

• Negative controls: SIGMAR1 knockout or knockdown samples (using CRISPR-Cas9 or siRNA technologies)

• Peptide competition assays: Co-incubating the antibody with specific SIGMAR1 antigen peptides to confirm binding specificity

• Cross-reactivity tests: Testing the antibody on multiple species if working with non-human models

Several studies have validated antibody specificity through the absence of staining in SIGMAR1 knockout mouse tissues, particularly in brain sections and dorsal root ganglion tissues .

What are the critical factors to consider when selecting a SIGMAR1 antibody for specific applications?

When selecting a SIGMAR1 antibody, consider:

• Target epitope: Antibodies raised against different regions of SIGMAR1 (N-terminal vs. C-terminal epitopes) may yield different results. Research has used antibodies raised against amino acid residues 52-69, 143-165, or full-length protein .

• Application compatibility: Validate that the antibody has been successfully used in your specific application (WB, IHC, IF, IP). For example, the Picoband antibody (A02493-2) has been validated for ELISA, IHC, and WB applications across human, monkey, mouse, and rat samples .

• Species reactivity: Ensure cross-reactivity with your experimental species. Some antibodies work across species (human, monkey, mouse, rat), while others are species-specific .

• Validation method: Prioritize antibodies validated using knockout/knockdown controls rather than just peptide blocking .

• Subcellular localization consistency: Confirm the antibody detects SIGMAR1 in expected subcellular compartments based on your biological question .

How can researchers validate SIGMAR1 antibody specificity in their experimental systems?

A multi-step validation approach is recommended:

• Western blot analysis: Confirm a single band at the expected molecular weight (~25 kDa for SIGMAR1) . Test multiple cell lines as demonstrated in the Picoband validation using HeLa, Caco-2, 293T, HepG2, A549, COLO-320, U-87MG, COS-7, mouse liver, and C2C12 samples .

• Genetic knockdown/knockout confirmation: Generate SIGMAR1 knockdown/knockout cells using siRNA, shRNA, or CRISPR-Cas9 approaches and confirm reduced or absent signal . For example, researchers achieved ~90% SIGMAR1 knockdown in SH-SY5Y cells using a lenti-vector expressing SIGMAR1 gRNA and dCas9-KRAB .

• Immunofluorescence co-localization: Co-stain with established ER markers to confirm expected localization patterns .

• Cross-platform validation: If possible, confirm findings using multiple detection methods (e.g., if using IHC, confirm with WB) .

• Compare antibody performance with published literature: Refer to established staining patterns to ensure consistency .

What are the most reliable SIGMAR1 antibody validation methods reported in the literature?

The most reliable validation methods include:

• CRISPR-Cas9 knockout validation: Several studies generated SIGMAR1 knockout cell lines (HEK293, NSC34) using CRISPR-Cas9 genome editing via lentiviral expression of Cas9 and SIGMAR1 gRNAs .

• siRNA/shRNA knockdown: Studies have validated antibodies using SIGMAR1 knockdown in SH-SY5Y cells (using dCas9-KRAB system) and oral cancer cell lines (SCC9, HN12) using shRNA .

• Knockout mouse tissues: Multiple studies used SIGMAR1 knockout mouse tissues as negative controls, particularly in immunohistochemistry of brain sections, dorsal root ganglion, and retina .

• Multi-cell line/tissue validation: Testing antibody performance across diverse samples, as demonstrated with the Picoband antibody, which was validated across 10 different cell/tissue samples .

• Co-localization with known SIGMAR1 interacting proteins: Validating antibody specificity through co-immunoprecipitation with known SIGMAR1-interacting proteins like IP3R3 .

What are the optimal immunohistochemistry protocols for detecting SIGMAR1 in fixed tissues?

Based on published methods:

• Tissue preparation:

  • Fix tissues in formalin and embed in paraffin

  • Section at 5 μm thickness

  • Deparaffinize and rehydrate sections

• Antigen retrieval:

  • Use antigen unmasking solution (e.g., Vector Laboratories H-3301)

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is effective for many tissue types

• Blocking steps:

  • Block endogenous peroxidases with 0.3% v/v hydrogen peroxide

  • Block non-specific binding with 5-10% serum (goat serum recommended)

• Primary antibody incubation:

  • Dilute SIGMAR1 antibody to 1:100-2 μg/ml (optimal concentration may vary by antibody)

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • Use appropriate HRP-conjugated secondary antibody (30-90 min at 37°C)

  • Amplify signal with Vectastain Elite Peroxidase Kit or similar

  • Visualize with DAB chromogen system

  • Counterstain with hematoxylin

• Example success: This protocol has been successfully applied to human adenocarcinoma of the right colon, human stomach cancer, rat colon, and mouse colon tissues .

What are the recommended protocols for Western blot detection of SIGMAR1?

Optimized Western blot protocol for SIGMAR1:

• Sample preparation:

  • Extract proteins using standard lysis buffers (RIPA or NP-40 based)

  • Load 30 μg protein per lane under reducing conditions

• Gel electrophoresis:

  • Use 5-20% gradient SDS-PAGE for optimal resolution

  • Run at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

• Transfer conditions:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Blocking:

  • Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody:

  • Dilute SIGMAR1 antibody to 0.25-1 μg/ml in blocking buffer

  • Incubate overnight at 4°C

• Washing and secondary antibody:

  • Wash with TBS-0.1% Tween (3 × 5 minutes)

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1.5 hours at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) detection system

  • Expected band size: ~25 kDa

• Validated cell lines: This protocol has been successfully used with human cell lines (HeLa, Caco-2, 293T, HepG2, A549, COLO-320, U-87MG), monkey (COS-7), and mouse (C2C12, liver) samples .

What are the optimal conditions for immunocytochemistry of SIGMAR1 in cultured cells?

Based on published protocols :

• Cell fixation:

  • Fix cells using 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.2% Triton X-100 (10 minutes)

• Blocking:

  • Block with 1-5% BSA or 5-10% normal serum in PBS (1 hour at room temperature)

• Primary antibody incubation:

  • Dilute SIGMAR1 antibody (typically 1:100-1:500) in blocking buffer

  • Co-stain with organelle markers (ER, mitochondria) for co-localization studies

  • Incubate overnight at 4°C

• Secondary antibody:

  • Use fluorophore-conjugated secondary antibodies appropriate for your microscopy setup

  • Incubate 1-2 hours at room temperature

  • Include DAPI for nuclear staining

• Imaging:

  • Confocal microscopy is recommended for accurate co-localization studies

  • Calculate Pearson's correlation coefficient for quantitative co-localization analysis

• Special considerations:

  • For agonist studies: treat cells with 10 mM (+)-Pentazocine or PRE-084 for 30 minutes before fixation

  • Some protocols require special treatment (0.02% SDS for 10 minutes) to visualize certain SIGMAR1 pools

How can researchers distinguish between different subcellular pools of SIGMAR1?

Distinguishing different SIGMAR1 pools requires specialized approaches:

• Subcellular fractionation combined with Western blotting:

  • Separate mitochondria, microsomes, MAM, nuclear, and plasma membrane fractions using differential centrifugation

  • Confirm fraction purity with organelle-specific markers (e.g., calnexin for ER, VDAC for mitochondria)

  • Probe for SIGMAR1 in each fraction

• High-resolution imaging techniques:

  • Super-resolution microscopy or immuno-electron microscopy provides more precise localization

  • Immuno-electron microscopy has revealed SIGMAR1 in nuclear envelope, subsurface ER cisternae, and ER-mitochondria contact sites

• Co-localization with compartment-specific markers:

  • ER markers: Sec61β, calnexin

  • MAM markers: FACL4, phospho-IP3R

  • Nuclear envelope: Lamin B1

  • Plasma membrane: Na+/K+ ATPase

  • Quantify co-localization using Pearson's or Mander's coefficients

• Fusion protein approaches:

  • GFP-tagged SIGMAR1 constructs with organelle-specific markers

  • APEX2-fusion constructs for electron microscopy visualization

  • Note: overexpression may alter natural distribution patterns

How should researchers interpret conflicting results regarding SIGMAR1 antibody staining patterns?

When facing conflicting staining patterns:

• Antibody epitope considerations:

  • Different antibodies target different epitopes and may expose different SIGMAR1 populations

  • Compare antibodies raised against N-terminal (aa 52-69) versus C-terminal (aa 143-165) regions

• Cell/tissue-specific expression patterns:

  • SIGMAR1 distribution varies by cell type and physiological state

  • Neuronal cells (NSC34, Neuro2a) show different patterns than non-neuronal cells (CHO, HEK293)

• Detection method sensitivity:

  • Immuno-electron microscopy provides higher resolution than immunofluorescence

  • Western blot of fractionated samples may detect populations missed by imaging

• Fixation and permeabilization artifacts:

  • Certain fixatives may mask epitopes or alter membrane structures

  • Compare paraformaldehyde versus methanol fixation

  • Some SIGMAR1 pools require detergent treatment (0.02% SDS) for visualization

• Resolution through multiple approaches:

  • Use complementary techniques (subcellular fractionation + imaging)

  • Compare endogenous versus tagged protein localization

  • Validate with SIGMAR1 knockout controls

What are the key considerations when studying SIGMAR1 translocation after agonist treatment?

When investigating SIGMAR1 translocation:

• Timing considerations:

  • Monitor time-dependent effects (30 minutes, 1 hour, 24 hours)

  • Some translocation events may be transient or biphasic

• Agonist selection and concentration:

  • Different agonists ((+)-Pentazocine, PRE-084) may produce different effects

  • Test multiple concentrations (typically 1-10 μM range)

  • Include both selective (PRE-084) and non-selective agonists for comparison

• Cell-type dependent responses:

  • SIGMAR1 dynamics appear cell-type dependent

  • Neuronal cells (Neuro2a) show different responses than non-neuronal cells

• Quantitative analysis:

  • Calculate co-localization coefficients before and after treatment

  • Use ratiometric measurements across multiple cellular compartments

  • Employ live-cell imaging when possible to track real-time dynamics

• Important negative findings:

  • Recent studies in Neuro2a cells found that SIGMAR1 did not translocate to PM, ER-PM contact sites, or nuclear envelope after agonist stimulation

  • Instead, SIGMAR1 may move within the ER structure rather than between organelles

How can researchers accurately quantify SIGMAR1 expression levels across different experimental conditions?

For accurate SIGMAR1 quantification:

• Western blot quantification:

  • Use appropriate housekeeping controls (β-actin is commonly used)

  • Apply densitometry analysis (ImageJ software is widely used)

  • Include standard curves with recombinant protein when possible

  • Compare relative expression levels across different samples

• qRT-PCR for mRNA quantification:

  • Design primers specific to SIGMAR1 transcript

  • Validate primer efficiency using standard curves

  • Use appropriate reference genes (multiple references preferred)

  • Calculate fold-change using 2^-ΔΔCT method

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity in defined regions

  • Calculate intensity ratios between compartments

  • Use software tools that account for background and cross-channel bleed-through

• Normalization strategies:

  • For knockdown studies, express as percentage relative to control

  • In tumor versus normal comparisons, normalize to matched normal tissue

  • For cross-species comparison, use conserved reference proteins

What experimental approaches can distinguish between SIGMAR1 expression and activation states?

To differentiate expression versus activation:

• Expression monitoring:

  • Total protein levels by Western blot

  • mRNA levels by qRT-PCR

  • Protein stability assays with cycloheximide chase

• Activation state markers:

  • SIGMAR1 oligomerization state (monomer vs. dimer on non-reducing gels)

  • Co-immunoprecipitation with client proteins (BiP/GRP78, IP3R3)

  • Subcellular redistribution from MAM to broader ER distribution

• Downstream signaling readouts:

  • Calcium signaling measurements

  • IP3R3 phosphorylation status

  • BDNF maturation and secretion

  • Phosphorylation of CREB (Ser133)

• Functional assays:

  • Mitochondrial function (membrane potential, respiration)

  • Autophagy flux (LC3-II, SQSTM1 levels)

  • ER stress markers (XBP1 splicing, CHOP expression)

• Pharmacological approach:

  • Compare agonist versus antagonist effects

  • Use selective ligands ((+)-pentazocine, pridopidine) versus non-selective (haloperidol)

  • Include appropriate vehicle controls

What is the current understanding of SIGMAR1 post-translational modifications and how can they be detected?

Current knowledge on SIGMAR1 post-translational modifications:

• Phosphorylation:

  • Predicted sites based on consensus sequences

  • Detection using phospho-specific antibodies

  • Phosphoproteomic analysis using mass spectrometry

  • Functional significance remains largely unexplored

• Glycosylation:

  • Limited evidence for glycosylation

  • Test with glycosidase treatment followed by Western blot mobility shift

• Oligomerization:

  • SIGMAR1 forms dimers and higher-order oligomers

  • Detect using non-reducing versus reducing SDS-PAGE

  • Crosslinking approaches can stabilize transient interactions

• Lipid modifications:

  • Predicted but not conclusively demonstrated

  • May affect membrane association and trafficking

• Ubiquitination:

  • May regulate SIGMAR1 turnover

  • Detect using immunoprecipitation followed by ubiquitin Western blot

  • Proteasome inhibitors can be used to stabilize ubiquitinated species

• Experimental approaches:

  • Mass spectrometry for unbiased modification profiling

  • Site-directed mutagenesis of modified residues

  • Pharmacological modulators of specific modifications

  • Correlation with functional outcomes