ALIS2 Antibody

What is Als2 protein and what is its significance in neurological research?

Als2 protein functions as a GTPase regulator that critically contributes to maintaining cellular homeostasis and membrane turnover in neuronal cells, thereby supporting nervous system integrity . By acting on cellular vesicle pathways, Als2 plays an essential role in controlling the survival and growth of spinal motoneurons . Understanding Als2 protein function is fundamental to investigating neurodegenerative conditions, particularly amyotrophic lateral sclerosis, where dysregulation of this protein may contribute to disease pathogenesis.

What types of Als2 antibodies are available for research applications?

Several validated Als2 antibodies are available for research applications, including:

• MAb 2-C6: A monoclonal antibody raised against the glycosylated form of Als2 protein with high specificity (KD of 8.2 ± 0.8 nM)

• MAb 2-C7: A monoclonal antibody raised against the Endo H-treated (deglycosylated) form of Als2

• Commercial antibodies such as ab170896: A rabbit recombinant monoclonal antibody suitable for multiple applications including IHC-P, IP, and WB techniques

These antibodies are typically IgG1 with kappa light chains and have been validated for specificity through various methods including Western blotting and immunolabeling experiments .

How are Als2 antibodies validated for research applications?

Rigorous validation of Als2 antibodies involves multiple complementary approaches:

What are optimal conditions for using Als2 antibodies in different applications?

Optimal conditions vary by application and antibody:

How should proper experimental controls be implemented when working with Als2 antibodies?

A robust experimental design requires multiple controls:

• Positive controls: Include samples with confirmed Als2 expression (e.g., SH-SY5Y, 293T, HeLa cells, human brain tissue)

• Negative controls:

  • Als2 knockdown/knockout samples (e.g., strain 2757 with near-zero Als2 expression)

  • Secondary antibody-only controls to detect non-specific binding

  • Isotype controls to identify non-specific binding due to antibody class

• Specificity controls: Include related proteins (other Als family members) to confirm antibody specificity

• Technical controls:

  • For Western blots: Include housekeeping proteins as loading controls

  • For binding kinetics: Include buffer blanks to correct for bulk shift and instrument noise

How can researchers troubleshoot weak or non-specific signals when using Als2 antibodies?

When encountering signal issues with Als2 antibodies, consider the following approaches:

For weak signals:

• Increase antibody concentration or extend incubation time

• Optimize antigen retrieval methods (heat-mediated retrieval with citrate buffer pH 6 for IHC-P)

• Verify target expression levels (even reintegration of Als2 promoter may not restore wild-type expression)

• Consider enzymatic treatment (some antibodies require Endo H treatment for signal visualization)

For non-specific signals:

• Select more specific antibodies (MAb 2-C6 showed better specificity than MAb 2-C7)

• Increase blocking duration and concentration

• Implement more stringent washing protocols

• Pre-adsorb antibodies with non-specific binding proteins

What is the significance of glycosylated versus deglycosylated antibodies in Als2 research?

The glycosylation state of Als2 immunogens significantly impacts antibody characteristics:

This difference demonstrates how post-translational modifications of immunogens affect epitope exposure during antibody development, potentially altering antibody specificity and cross-reactivity profiles .

How can binding kinetics data inform experimental design with Als2 antibodies?

Understanding binding kinetics provides crucial insights for optimizing experimental protocols:

• Selection criteria: Higher affinity antibodies (like MAb 2-C6 with KD of 8.2 ± 0.8 nM) may be preferred for detecting low-abundance targets

• Protocol optimization:

  • Association/dissociation rates determine optimal incubation and washing times

  • Endo H-treated Als2 completely dissociates after 10 minutes, requiring no regeneration protocol

  • Glycosylated Als2 requires regeneration with 10 mM glycine (pH 1.75) for 30 seconds

• Quantitative analysis:

  • Known binding constants enable mathematical modeling of antibody-antigen interactions

  • Chi-square values (e.g., 1.1 ± 0.6 for MAb 2-C6) indicate goodness of fit for kinetic models

What methodological approaches are recommended for studying Als2 protein localization in neuronal tissues?

For optimal Als2 localization studies in neuronal tissues:

• Sample preparation:

  • Use fresh tissues or properly fixed samples (paraffin-embedded human brain tissue works well)

  • Perform heat-mediated antigen retrieval with citrate buffer pH 6 before IHC staining

• Antibody selection and dilution:

  • Use validated Als2-specific antibodies like ab170896 at 1/50 dilution for IHC-P

  • Consider knockout-validated antibodies to ensure specificity

• Visualization strategy:

  • Implement appropriate detection systems based on expression levels

  • Consider that Als2 expression may vary significantly between cell types and conditions

  • Note that expression patterns may be punctate in some cells and diffuse in others

• Controls integration:

  • Include tissues with known Als2 expression patterns

  • Compare with Als2-deficient samples when available

  • Use additional markers to correlate Als2 localization with subcellular compartments

How can Als2 antibodies contribute to understanding ALS pathogenesis?

Als2 antibodies enable multiple research approaches for investigating ALS mechanisms:

• Expression profiling: Quantify Als2 protein levels in affected versus unaffected tissues using Western blotting with antibodies like ab170896

• Localization studies:

  • Examine Als2 distribution changes during disease progression using IHC-P

  • Investigate redistribution between cellular compartments that may indicate pathological changes

• Protein interaction analysis:

  • Use immunoprecipitation to identify Als2 binding partners that may be disrupted in disease

  • Compare interaction networks between normal and pathological conditions

• Functional studies:

  • Employ Als2 antibodies to block protein function in cellular models

  • Investigate effects on cellular homeostasis and membrane turnover pathways

What considerations are important for multiplexing Als2 antibodies with other neuronal markers?

Successful multiplex immunostaining with Als2 antibodies requires careful planning:

• Antibody compatibility:

  • Select primary antibodies from different species or isotypes (Als2 antibodies include rabbit recombinant monoclonal or mouse IgG1 options)

  • Test for cross-reactivity between detection systems

• Protocol optimization:

  • Ensure antigen retrieval methods preserve all target epitopes

  • Determine if sequential staining is necessary to avoid interference

  • Optimize each antibody individually before combining

• Signal detection:

  • Choose fluorophores with minimal spectral overlap

  • Consider that Als2 may require signal amplification due to variable expression levels

  • Include single-stained controls for each antibody for proper signal separation

What quantitative approaches can be implemented to measure Als2 protein expression levels accurately?

For precise quantification of Als2 protein:

• Western blot quantification:

  • Use validated antibodies like ab170896 at 1/1000 dilution

  • Implement appropriate housekeeping protein controls

  • Employ digital image analysis with standard curves for absolute quantification

• Flow cytometry:

  • Optimize fixation and permeabilization protocols for intracellular Als2 detection

  • Use median fluorescence intensity for comparative analysis

  • Include calibration beads for standardization

• Mass spectrometry validation:

  • Complement antibody-based quantification with MS-based proteomics

  • Consider targeted approaches like selected reaction monitoring for low-abundance detection

  • Correlate antibody-based and MS-based quantification for validation

• Real-time quantitative considerations:

  • Reference qRT-PCR data (e.g., Als2 transcript copy numbers: 28615 ± 3510 in wild-type vs. 33 ± 11 in knockdown)

  • Correlate transcript levels with protein expression to understand regulatory mechanisms