ATL1 Antibody, HRP conjugated

What is ATL1 antibody and what cellular functions does ATL1 target?

ATL1 antibody targets Atlastin-1 (also known as SPG3A), a GTPase that plays crucial roles in cellular membrane dynamics. Specifically, ATL1 functions in endoplasmic reticulum tubular network biogenesis through the formation of trans-homooligomers and mediates homotypic fusion of endoplasmic reticulum membranes. The protein may also regulate Golgi biogenesis and axonal development .

When selecting an ATL1 antibody for research applications, it's important to verify epitope specificity, as some antibodies are specifically designed not to cross-react with related family members ATL2 and ATL3 . For experimental design, consider that ATL1 is expressed predominantly in the central nervous system and has been implicated in hereditary spastic paraplegia, making it particularly relevant for neuroscience research applications.

What are the advantages of using HRP-conjugated antibodies in immunoassays?

HRP-conjugated antibodies offer several significant methodological advantages in research applications:

• Direct detection capability eliminates the need for secondary antibody incubation, reducing experiment time and potential sources of variability

• Signal amplification through enzymatic activity enables detection of low-abundance targets

• Compatibility with multiple substrates (colorimetric, chemiluminescent, fluorescent) provides flexibility across experimental platforms

• Reduced background noise in properly optimized systems

• Enhanced sensitivity for detecting low levels of target protein

Research demonstrates that optimized HRP-antibody conjugates can detect antigens at concentrations as low as 1.5 ng in properly designed assays . This sensitivity makes HRP-conjugated antibodies particularly valuable for detecting proteins like ATL1 that may be expressed at modest levels in certain cell types or under specific conditions.

How should ATL1 antibody, HRP conjugated be stored to maintain optimal activity?

Proper storage is critical for maintaining both the antigen-binding capacity of the ATL1 antibody portion and the enzymatic activity of the HRP component. The recommended protocol includes:

• Store at -20°C for long-term storage in small aliquots (typically 10-20 μL)

• Avoid repeated freeze-thaw cycles that can denature both antibody and enzyme components

• For working solutions, store at 4°C for no longer than 1-2 weeks

• Include carrier proteins (e.g., 0.1-1% BSA) in storage buffers if not already in the formulation

• Protect from light exposure, particularly if storing in clear tubes

• Avoid oxidizing environments and contamination with heavy metals that can inactivate HRP

Implementing these measures will help prevent activity loss that commonly occurs through improper handling. Recombinant antibody-HRP conjugates may show enhanced stability compared to chemically conjugated versions due to their defined stoichiometry and structural consistency .

What applications are most suitable for ATL1 antibody, HRP conjugated?

ATL1 antibody, HRP conjugated is particularly well-suited for several research applications:

• Western blotting (WB): Provides direct detection of ATL1 protein (~63 kDa) without secondary antibody incubation. Optimize dilutions typically between 1:1000-1:5000 depending on protein abundance and conjugation quality.

• Immunohistochemistry on paraffin sections (IHC-P): Enables visualization of ATL1 in fixed tissue sections. Critical considerations include antigen retrieval optimization and endogenous peroxidase blocking.

• Immunocytochemistry/Immunofluorescence (ICC/IF): Allows subcellular localization studies, particularly valuable for examining ATL1's distribution in the endoplasmic reticulum network.

• Flow cytometry (intracellular): Permits quantification of ATL1 expression across cell populations, though requires proper permeabilization protocols.

The choice of application should be guided by experimental objectives, with Western blotting often providing the most straightforward initial validation of antibody specificity before proceeding to more complex localization studies .

What controls should be incorporated when using ATL1 antibody, HRP conjugated?

Rigorous experimental design requires appropriate controls to validate results obtained with ATL1 antibody, HRP conjugated:

• Positive tissue control: Include brain or spinal cord tissue known to express ATL1 at detectable levels

• Negative controls:

  • Tissues known not to express ATL1

  • ATL1 knockout or knockdown samples (when available)

  • Primary antibody omission control to assess background from detection system

• Specificity controls:

  • Peptide competition/neutralization assay with immunizing peptide

  • Isotype control (same species and isotype with irrelevant specificity)

  • Cross-reactivity assessment with ATL2/ATL3 to confirm specificity

• Technical controls:

  • Endogenous peroxidase blocking validation

  • System control (substrate only) to assess detection system background

  • Loading controls for Western blotting (β-actin, GAPDH, total protein stain)

Implementing these controls systematically enables confident interpretation of ATL1 detection patterns and strengthens the validity of research findings.

How does the conjugation process affect the sensitivity and specificity of ATL1 antibody detection?

The conjugation process between HRP and antibodies significantly impacts assay performance through several mechanisms:

• Conjugation chemistry: The most common approach uses periodate oxidation to generate aldehyde groups on HRP's carbohydrate moieties that react with primary amines on antibodies . This preserves antibody binding sites better than methods targeting amino acids directly involved in antigen recognition.

• Enzyme-to-antibody ratio: Optimal ratios vary by application, with higher ratios increasing sensitivity but potentially reducing specificity if HRP sterically hinders antigen binding.

• Enhanced methods: Lyophilization of activated HRP before conjugation significantly improves efficiency by concentrating reactants without changing their amounts, leading to conjugates effective at dilutions of 1:5000 versus only 1:25 for conventional methods .

• Preservation of functional domains: Successful conjugation maintains both HRP enzymatic activity and antibody binding capacity, with recombinant approaches potentially offering advantages through defined attachment points .

Research demonstrates that lyophilization-enhanced conjugation can improve detection sensitivity up to 200-fold compared to classical methods, with conjugates capable of detecting antigens at concentrations as low as 1.5 ng .

What protocols yield the best results for Western blotting using ATL1 antibody, HRP conjugated?

Optimized Western blotting protocols for ATL1 antibody, HRP conjugated require attention to several key parameters:

• Sample preparation:

  • Use RIPA or NP-40 lysis buffers with protease inhibitors

  • Include reducing agents in sample buffer to break disulfide bonds

  • Heat samples at 95°C for 5 minutes in Laemmli buffer

• Electrophoresis and transfer:

  • Use 10% SDS-PAGE for optimal resolution of ATL1 (~63 kDa)

  • Transfer to PVDF membrane (more protein binding capacity than nitrocellulose)

  • Verify transfer efficiency with reversible protein stain before blocking

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST (0.1% Tween-20)

  • Dilute ATL1 antibody, HRP conjugated 1:1000-1:5000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Wash extensively (5 × 5 minutes) with TBST

• Detection optimization:

  • Select substrate based on expected abundance (standard ECL for moderate expression, enhanced chemiluminescence for low abundance)

  • For quantitative analysis, use digital imaging systems with linear dynamic range

  • Include molecular weight markers to confirm expected size

• Troubleshooting high background:

  • Increase washing stringency (more washes, higher detergent concentration)

  • Further dilute antibody

  • Try alternative blocking reagents (BSA, commercial blockers)

Research on enhanced conjugation methods indicates significantly improved sensitivity, potentially allowing detection of low ATL1 expression levels that might be missed with conventional conjugates .

How can researchers optimize immunohistochemistry protocols with ATL1 antibody, HRP conjugated?

Successful immunohistochemistry with ATL1 antibody, HRP conjugated requires systematic optimization:

• Tissue preparation considerations:

  • Fixation: 10% neutral buffered formalin for 24-48 hours optimal

  • Sectioning: 4-5 μm thickness recommended

  • Storage: freshly cut sections preferred over stored slides

• Critical pretreatment steps:

  • Deparaffinization must be complete (extended xylene incubation if needed)

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) typically most effective

  • Endogenous peroxidase blocking: 0.3% H₂O₂ in methanol for 30 minutes

• Staining protocol optimization:

  • Blocking: 5-10% normal serum in PBS for 60 minutes

  • Primary antibody: Titrate ATL1 antibody, HRP conjugated (typically 1:50-1:200)

  • Incubation: Overnight at 4°C in humidified chamber

  • Washing: PBS with 0.05% Tween-20, 3 × 5 minutes

• Detection refinement:

  • DAB development: Monitor microscopically to optimize signal-to-noise ratio

  • Counterstaining: Light hematoxylin counterstain to avoid obscuring specific signal

  • Controls: Include run-to-run standardization controls

• Special considerations for ATL1:

  • Neural tissues require careful blocking of lipid-rich regions

  • Double staining with ER markers may help confirm specificity

  • Tyramide signal amplification can enhance detection in low-expression samples

This methodological approach enables reliable visualization of ATL1 in tissues while minimizing background and non-specific staining artifacts.

What are the optimal lyophilization conditions for enhancing ATL1-HRP conjugation?

Lyophilization of activated HRP represents a significant methodological advancement for improving conjugation efficiency. The optimized protocol includes:

• HRP activation process:

  • Dissolve HRP in distilled water (typically 4 mg/ml)

  • Add sodium meta-periodate (5-8 mM final concentration)

  • Incubate for 30 minutes at room temperature in the dark

  • Purify by gel filtration or dialysis to remove excess periodate

• Lyophilization parameters:

  • Pre-freeze activated HRP solution at -80°C for 2 hours

  • Lyophilize at -40°C to -50°C under vacuum (typically 0.1 mbar)

  • Include cryoprotectants like 1% trehalose if stability is a concern

  • Store lyophilized activated HRP at -20°C

• Conjugation with ATL1 antibody:

  • Reconstitute lyophilized HRP with minimal volume of carbonate buffer (pH 9.5)

  • Add ATL1 antibody at 1 mg/ml concentration

  • Maintain 1:4 to 1:6 molar ratio of antibody:HRP

  • Incubate 2-3 hours at room temperature

  • Add sodium borohydride to stabilize conjugate

• Purification considerations:

  • Use size exclusion chromatography to separate conjugates from free HRP/antibody

  • Dialyze against PBS with preservatives

  • Confirm conjugation by spectrophotometric analysis

Research demonstrates this approach can improve detection sensitivity up to 200-fold compared to conventional methods, with conjugates remaining effective at dilutions of 1:5000 versus only 1:25 for standard conjugation protocols .

How can researchers address contradictory results between ATL1 antibody detection and mRNA expression data?

Discrepancies between protein detection using ATL1 antibody, HRP conjugated and corresponding mRNA data require systematic investigation:

• Technical validation approaches:

  • Verify antibody specificity through Western blotting of recombinant ATL1

  • Confirm ATL1 knockdown reduces antibody signal proportionally

  • Test multiple antibodies against different ATL1 epitopes

  • Examine detection using alternative methods (IF vs. WB vs. IHC)

• Biological explanations to investigate:

  • Post-transcriptional regulation (miRNAs targeting ATL1 mRNA)

  • Differences in protein vs. mRNA stability and turnover rates

  • Translational efficiency factors affecting protein synthesis

  • Post-translational modifications potentially masking antibody epitopes

• Experimental design considerations:

  • Ensure temporal alignment between protein and mRNA sampling

  • Account for cell type heterogeneity in complex tissues

  • Consider subcellular compartmentalization affecting extraction efficiency

  • Evaluate mRNA splicing variants potentially not detected by antibody

• Integrated analysis strategies:

  • Correlate protein and mRNA data across larger sample sets

  • Implement absolute quantification methods for both protein and mRNA

  • Consider proteogenomic approaches integrating mass spectrometry with RNA-seq

  • Examine patterns across developmental or disease progression timepoints

These methodological approaches help determine whether discrepancies represent technical artifacts or biologically meaningful regulatory mechanisms affecting ATL1 expression.

How does substrate selection impact the sensitivity of ATL1 antibody, HRP conjugated detection?

Substrate selection critically influences detection sensitivity and signal characteristics when using ATL1 antibody, HRP conjugated:

• Colorimetric substrates:

  • DAB (3,3'-diaminobenzidine): Produces brown precipitate; most stable for archival samples but least sensitive

  • TMB (3,3',5,5'-tetramethylbenzidine): Produces blue color; higher sensitivity for ELISA but less stable

  • AEC (3-amino-9-ethylcarbazole): Produces red product; soluble in alcohol, requires aqueous mounting

• Chemiluminescent substrates:

  • Standard ECL: Suitable for moderate ATL1 expression levels

  • Enhanced ECL (with phenols or luminol derivatives): 10-50× more sensitive

  • SuperSignal/Femto substrates: Highest sensitivity (200-1000× standard ECL)

• Tyramide-based substrates:

  • Enable signal amplification through tyramide deposition

  • Permit fluorescent detection when coupled with appropriate fluorophores

  • Allow multiplexing with other targets after HRP inactivation

The table below summarizes relative sensitivities for detecting ATL1:

For ATL1 detection in tissues or cells with low expression levels, enhanced chemiluminescent or tyramide-based substrates provide optimal sensitivity .

What dilution optimization strategies are recommended for ATL1 antibody, HRP conjugated?

Determining optimal dilution for ATL1 antibody, HRP conjugated requires systematic optimization:

• Titration approach:

  • Prepare serial dilutions (1:50, 1:100, 1:500, 1:1000, 1:5000)

  • Test against samples with known ATL1 expression

  • Evaluate signal-to-noise ratio rather than absolute signal intensity

  • Select dilution providing specific signal with minimal background

• Application-specific considerations:

  • Western blotting: Start with higher dilutions (1:1000-1:5000)

  • IHC/ICC: Begin with lower dilutions (1:50-1:200)

  • ELISA: Requires separate optimization for coating vs. detection

• Optimization protocol:

  • Prepare master dilution series using identical buffer composition

  • Process all dilutions simultaneously under identical conditions

  • Include appropriate positive and negative controls

  • Document results quantitatively when possible

• Special considerations for enhanced conjugates:

  • Lyophilization-enhanced HRP conjugates may remain effective at dilutions up to 1:5000

  • Conventional conjugates typically require more concentrated preparations (1:25-1:100)

  • Substrate choice influences optimal dilution significantly

Research demonstrates that optimized conjugation methods can dramatically improve antibody efficiency, with enhanced conjugates showing effective detection at 200× more dilute concentrations than conventional conjugates .

What troubleshooting approaches are recommended for non-specific binding issues with ATL1 antibody, HRP conjugated?

Non-specific binding can significantly impact experimental results with ATL1 antibody, HRP conjugated. A systematic troubleshooting approach includes:

• Blocking optimization:

  • Test alternative blocking reagents (BSA, casein, commercial blockers)

  • Extend blocking time (2 hours at room temperature)

  • Include mild detergents (0.1-0.3% Triton X-100) for membrane permeabilization

  • Consider additives like 5% normal serum from the same species as tissue origin

• Antibody dilution refinement:

  • Further dilute primary antibody (may require 2-5× higher dilution)

  • Prepare antibody in fresh blocking buffer containing 0.05% Tween-20

  • Consider overnight incubation at 4°C instead of shorter room temperature incubation

  • Pre-absorb antibody with tissue homogenates from species of interest

• Washing protocol enhancement:

  • Increase wash buffer detergent concentration (0.1% Tween-20)

  • Extend washing times (5-6 washes at 5-10 minutes each)

  • Use higher salt concentration in wash buffer (up to 500 mM NaCl)

  • Consider adding 0.2M glycine (pH 2.5) wash to remove weakly bound antibodies

• Sample-specific approaches:

  • For tissues with high endogenous peroxidase, double peroxidase quenching step

  • For tissues with biotin content, implement avidin-biotin blocking

  • For highly autofluorescent samples, use Sudan Black B treatment

  • Consider antigen retrieval modification if epitope accessibility is suspected

These methodological refinements can significantly improve signal-to-noise ratio and ensure that observed signals accurately represent ATL1 distribution .

What are the considerations for multiplexing experiments involving ATL1 antibody, HRP conjugated?

Multiplexing with ATL1 antibody, HRP conjugated requires careful experimental design to avoid signal interference:

• Sequential detection strategies:

  • Complete first HRP detection cycle with substrate development

  • Inactivate HRP using 0.3% hydrogen peroxide (10-15 minutes)

  • Apply heat treatment (microwave: 10 minutes in citrate buffer) to strip antibodies

  • Verify complete inactivation/stripping with substrate-only control

  • Proceed with second antibody-HRP conjugate detection

• Tyramide signal amplification (TSA) approach:

  • Use dilute ATL1 antibody, HRP conjugated with fluorescent tyramide substrate

  • Permanent signal deposition allows complete HRP inactivation

  • Apply second HRP-conjugated antibody targeting different protein

  • Use different fluorophore-labeled tyramide for second target

  • Capture separate fluorescence channels without signal overlap

• Complementary detection systems:

  • Combine HRP-conjugated ATL1 antibody with alkaline phosphatase-conjugated antibodies

  • Use chromogenic substrates with distinct colors (brown DAB for ATL1, red Fast Red for second target)

  • For fluorescence, combine HRP-TSA system with directly labeled fluorescent antibodies

• Advanced considerations:

  • Include comprehensive controls for each target individually

  • Account for potential epitope masking in dense target regions

  • Consider target abundance differences when designing sequential detection

  • Implement spectral unmixing for fluorescent multiplexing applications

These methodological approaches enable simultaneous visualization of ATL1 with other proteins of interest, particularly valuable for co-localization studies with other ER-associated proteins or interaction partners.

How should quantitative data from ATL1 antibody, HRP conjugated experiments be normalized?

Proper normalization is essential for reliable quantitative analysis of ATL1 expression:

• Western blot densitometry normalization:

  • To housekeeping proteins: Calculate ATL1 signal relative to β-actin, GAPDH, or tubulin

  • To total protein: Use Ponceau S, SYPRO Ruby, or stain-free technology for loading control

  • Correction method: Divide ATL1 band intensity by normalization control intensity

  • Data presentation: Report as fold-change relative to control condition

• Immunohistochemistry quantification methods:

  • H-score approach: Multiply percentage of positive cells by staining intensity (0-3)

  • Automated image analysis: Measure DAB optical density and normalize to tissue area

  • Cell counting method: Calculate percentage of ATL1-positive cells per field

  • Relative intensity measurement: Compare target region to internal reference region

• ELISA data normalization:

  • Standard curve method: Use recombinant ATL1 protein standards

  • Curve fitting: Apply 4-parameter logistic regression for accurate interpolation

  • Plate normalization: Include control sample across multiple plates

  • Dilution normalization: Account for any sample dilution factors

• Statistical processing:

  • Apply appropriate tests based on data distribution (parametric vs. non-parametric)

  • Account for technical and biological replicates in statistical model

  • Report variability consistently (standard deviation or standard error)

  • Provide complete information on sample size and statistical methodology

Enhanced conjugation methods, such as those using lyophilization, can improve detection sensitivity to as low as 1.5 ng of antigen, enabling more accurate quantification of ATL1 across a wider dynamic range .

What are the limitations of ATL1 antibody, HRP conjugated in cross-species applications?

Cross-species application of ATL1 antibody, HRP conjugated requires careful consideration of several limitations:

• Epitope conservation issues:

  • Human ATL1 shares approximately 94% amino acid identity with mouse and 93% with rat

  • Specific epitope regions may have lower conservation

  • Antibodies raised against human ATL1 may show variable cross-reactivity

  • Terminal regions typically show lower conservation than functional domains

• Validation requirements for cross-species use:

  • Western blotting should confirm single band of expected molecular weight

  • Positive controls from target species are essential

  • Consider using knockout/knockdown models as negative controls

  • Peptide competition assays with species-specific peptides provide additional validation

• Technical adjustments for non-target species:

  • Higher antibody concentrations typically required (2-5× higher)

  • Modified blocking buffers may be necessary (species-matched serum)

  • Extended incubation times often improve signal detection

  • Antigen retrieval optimization is particularly important for cross-species IHC

• Alternative approaches when cross-reactivity is insufficient:

  • Use unconjugated primary antibody with species-specific HRP-secondary antibody

  • Consider custom antibody generation against conserved epitopes

  • Utilize recombinant expression systems with tagged ATL1 from species of interest

  • Implement genetic models with reporter-tagged endogenous ATL1

These methodological considerations ensure appropriate interpretation of cross-species data and prevent misattribution of signals when working with ATL1 antibody, HRP conjugated in non-human models.

How should researchers interpret variations in ATL1 staining patterns across different cell compartments?

ATL1 (Atlastin-1) primarily localizes to the endoplasmic reticulum, but variations in staining patterns require careful interpretation:

These methodological approaches ensure that observed variations in ATL1 localization are biologically meaningful rather than technical artifacts, particularly important when studying potential pathological changes in ATL1 distribution in disease models.

What statistical approaches are appropriate for analyzing ATL1 expression across different tissue samples?

Selection of appropriate statistical methods for ATL1 expression analysis depends on experimental design and data characteristics:

• For two-group comparisons:

  • Student's t-test for normally distributed data with equal variances

  • Welch's t-test for normally distributed data with unequal variances

  • Mann-Whitney U test for non-parametric comparison of ranks

  • Paired t-test for matched sample designs

• For multiple group comparisons:

  • One-way ANOVA with post-hoc tests (Tukey or Bonferroni) for normally distributed data

  • Kruskal-Wallis with Dunn's post-test for non-parametric multiple comparisons

  • Two-way ANOVA for experiments with two independent variables

  • Mixed models for repeated measures with missing data points

• For correlation analysis:

  • Pearson correlation for linear relationships between normally distributed variables

  • Spearman rank correlation for non-parametric or non-linear relationships

  • Multiple regression for identifying predictive variables of ATL1 expression

  • Hierarchical clustering to identify patterns across multiple markers

• Advanced considerations for ATL1 studies:

  • Account for technical variation through nested designs

  • Consider weighted analysis if variance is heterogeneous

  • Implement multiple comparison correction for large-scale studies

  • Use bootstrapping approaches for small sample sizes

Statistical significance in ATL1 expression studies is typically set at p < 0.05, though more stringent thresholds may be appropriate for exploratory or large-scale analyses to control false discovery rates.

How can researchers optimize signal-to-noise ratios when using ATL1 antibody, HRP conjugated?

Maximizing signal-to-noise ratio is critical for detecting specific ATL1 signals above background:

• Western blotting optimization:

  • Use PVDF membranes for higher protein binding capacity

  • Implement gradient SDS-PAGE for better separation near ATL1's molecular weight

  • Optimize transfer conditions (time, buffer composition, voltage)

  • Use milk-based blocking rather than BSA for HRP systems

  • Apply optimized washing protocols (increased number and duration)

• IHC/ICC signal enhancement:

  • Test multiple antigen retrieval methods systematically

  • Implement dual endogenous peroxidase blocking steps

  • Use signal amplification systems for low abundance targets

  • Optimize primary antibody concentration through titration

  • Apply polymer detection systems for enhanced sensitivity

• ELISA optimization:

  • Use high-binding microplates for maximum protein capture

  • Implement optimized blocking to prevent non-specific binding

  • Consider kinetic reading to determine optimal signal collection time

  • Use reference standards on each plate for inter-assay normalization

  • Apply optimal substrate development times based on positive controls

• General principles for improved signal-to-noise:

  • Increase antibody incubation time while decreasing concentration

  • Optimize detergent concentration in wash buffers

  • Use fresh reagents, particularly substrates

  • Include appropriate negative controls for background assessment

  • Consider signal amplification methods for low-expression samples

Research on enhanced conjugation methods demonstrates that lyophilization-enhanced HRP-antibody conjugates can provide significantly improved signal-to-noise ratios, potentially detecting antigen at concentrations as low as 1.5 ng with minimal background .