AZS22-14 Antibody

What is AZS22-14 Antibody and what target protein does it recognize?

AZS22-14 Antibody (catalog code CSB-PA191061XA01ZAX) is a custom antibody that recognizes a protein in Zea mays (maize) with UniProt accession number Q94IM1 . Based on nomenclature patterns in the literature, AZS22-14 likely targets a 22 kDa alpha-zein storage protein, as similar naming conventions are used for other zein proteins in maize . This antibody is part of Cusabio's custom antibody collection, which includes several other antibodies targeting various zein proteins .

How does AZS22-14 Antibody relate to active zone (AZ) research?

While the antibody name contains "AZ," this should not be confused with antibodies targeting neuronal active zone (AZ) proteins. In neuroscience research, active zone antibodies are critical for studying presynaptic release sites where synaptic vesicles (SVs) dock and fuse . The AZS22-14 designation more likely refers to Alpha-Zein Storage protein of 22 kDa, as opposed to neuronal active zone scaffold proteins like BRP, RIM-BP, or Unc13A that are studied using different antibodies .

What are the recommended experimental applications for AZS22-14 Antibody?

While specific validation data is not provided in the search results, zein protein antibodies are typically optimized for Western blotting, immunohistochemistry, and immunoprecipitation applications . When planning experiments, researchers should follow protocols similar to those used with other plant storage protein antibodies, which typically include protein extraction under reducing conditions, SDS-PAGE separation, and standard immunodetection methods.

What protocols should be followed for immunohistochemistry with AZS22-14 Antibody?

For optimal immunohistochemical analysis with plant tissue antibodies, follow this methodology adapted from established protocols:

• Collect and dissect the tissue of interest (e.g., maize endosperm)

• Post-fix with 2% paraformaldehyde in PBS for 20 minutes at room temperature

• Cryoprotect in 20% sucrose/PBS at 4°C overnight

• Embed in optimal cutting temperature compound

• Cut sections (25-μm thick) using a cryostat

• Block with 2% bovine serum albumin, 2% normal goat serum, and 0.1% Triton X-100 in PBS

• Incubate with AZS22-14 primary antibody at 4°C overnight

• Wash with PBS and incubate with appropriate fluorophore-conjugated secondary antibody

• Mount in antifade reagent with a No. 1.5 cover glass

This approach has been validated for detecting protein expression in plant tissues and should provide reliable results with AZS22-14 Antibody.

How should protein extraction be optimized for Western blotting with AZS22-14 Antibody?

For effective Western blotting of zein proteins, extract proteins using a method similar to this maize seed extraction protocol:

• Grind seeds or tissue in liquid nitrogen to a fine powder

• Extract with buffer containing: 150 mM NaCl, 50 mM Tris-HCl (pH 7.4-7.5), 2.5 mM EDTA, 0.2% NP-40, 2.5 mM PMSF, and 1% protease inhibitor cocktail

• Determine protein concentration by Bradford method

• Separate proteins by SDS-PAGE under reducing conditions

• Transfer to membrane and block with appropriate blocking buffer

• Incubate with AZS22-14 primary antibody, followed by appropriate HRP-conjugated secondary antibody

• Develop using a chemiluminescent detection system

When interpreting results, note that the apparent molecular weight of plant proteins on SDS-PAGE may differ from theoretical weights due to post-translational modifications .

How can I quantify protein expression levels using AZS22-14 Antibody?

For accurate quantification of immunostaining intensity:

• Obtain images using consistent microscope settings (laser intensity, zoom, and detector gain)

• Process paired samples (experimental and control) on the same slide and stain together

• Perform image analysis using ImageJ or similar software:

  • Generate a binary mask of the region of interest

  • Determine average intensity within the mask for each sample

  • For Western blots, use densitometry to measure band intensity relative to loading controls

• For studying protein distribution beyond the primary region, measure signal in an annulus surrounding the original mask

This approach allows for reliable comparison of protein expression levels between experimental conditions.

How can I validate the specificity of AZS22-14 Antibody in my experiments?

To confirm antibody specificity:

• Compare staining patterns with known expression patterns of the target protein

• Include positive controls (tissues known to express the target) and negative controls (tissues without expression or with the primary antibody omitted)

• Perform peptide competition assays to confirm binding specificity

• For zein proteins specifically, compare results with the amino acid composition table below to verify expected properties:

What might cause unexpected banding patterns in Western blots with AZS22-14 Antibody?

When troubleshooting unexpected results:

• Consider that the apparent molecular weight of plant proteins can differ significantly from their theoretical weight (e.g., ZmO2 has a predicted molecular weight of 47.075 kDa but migrates at approximately 68-72 kDa on SDS-PAGE)

• Evaluate potential post-translational modifications affecting mobility

• Check for protein aggregation or oligomerization, particularly with storage proteins like zeins

• Verify extraction buffer compatibility with your target protein

• Test different reducing conditions, as disulfide bonds in storage proteins can affect migration patterns

• Consider cross-reactivity with similar proteins, especially in the zein/kafirin family which shares structural similarities

How can I distinguish between non-specific binding and true signal in immunohistochemistry?

To differentiate specific from non-specific signals:

• Include multiple controls:

  • Tissue from knockout or knockdown lines if available

  • Preabsorption of antibody with purified antigen

  • Secondary antibody-only controls

• Compare staining patterns with in situ hybridization data for the target gene

• Use confocal microscopy with appropriate filter settings to minimize autofluorescence from plant tissues

• Compare localization patterns with known subcellular distributions of similar proteins

• For maize seed sections, be particularly aware of autofluorescence from cell walls and natural pigments

How can AZS22-14 Antibody be used in studying transgenic events affecting zein expression?

For transgenic research applications:

• Design experiments similar to studies of kafirin gene clusters in maize, where protein extraction and isoelectric focusing (IEF) protein gel electrophoresis were used to distinguish transgenic kafirin proteins from endogenous zeins

• Extract prolamins from transgenic and non-transgenic seeds as controls

• Use Western blotting with AZS22-14 to detect changes in target protein expression

• For validation, complement antibody detection with gene expression analysis using RT-PCR or RNA-seq

• Track protein expression through multiple generations to assess genetic stability of the transgene

This approach has been successful in identifying and characterizing novel protein expression in transgenic maize lines.

How might AZS22-14 Antibody be used to study protein-protein interactions?

For investigating protein interactions:

• Use co-immunoprecipitation (Co-IP):

  • Extract proteins from maize tissues using a buffer that preserves protein-protein interactions

  • Immunoprecipitate using AZS22-14 Antibody coupled to protein A/G beads

  • Analyze precipitated complexes by SDS-PAGE and mass spectrometry to identify interacting partners

• Alternatively, perform GST pull-down assays:

  • Express potential interacting proteins as GST fusion proteins

  • Incubate with maize seed protein extracts

  • Detect interactions using Western blotting with AZS22-14 Antibody

• Validate interactions using yeast two-hybrid or bimolecular fluorescence complementation assays

These approaches would identify proteins that interact with the AZS22-14 target in vivo.

How can AZS22-14 Antibody contribute to understanding zein gene regulation?

For regulatory studies:

• Compare protein expression patterns with transcriptional data to identify post-transcriptional regulation

• Analyze protein levels in different genetic backgrounds, particularly those with mutations in transcription factors like Opaque-2 (O2)

• Investigate temporal expression during seed development (3-36 days after pollination) to establish developmental regulation patterns

• Examine response to environmental stresses or nutritional conditions

• Study potential interactions with regulatory proteins such as ZmTaxilin, which has been shown to negatively regulate zein gene expression

This research would provide insights into the complex regulatory networks controlling storage protein accumulation in maize endosperm.

How can AZS22-14 be used in comparative studies of cereal storage proteins?

For comparative cereal protein research:

• Extract prolamins from different cereals (maize, sorghum, rice) for side-by-side comparison

• Use Western blotting and IEF to compare electrophoretic mobility patterns

• Assess cross-reactivity of AZS22-14 with homologous proteins in related species

• Compare localization patterns in seed tissues across species

• Study evolutionary relationships between zein, kafirin, and other prolamin families based on antibody cross-reactivity and amino acid composition differences

This research would illuminate evolutionary relationships and functional conservation among cereal storage proteins.

How might AZS22-14 Antibody be applied in studies of protein quality improvement in maize?

For protein quality improvement research:

• Use AZS22-14 to monitor changes in zein profiles in high-lysine or modified-protein maize varieties

• Analyze how manipulating regulatory factors affects the balance between zein subtypes

• Study how environmental conditions affect zein accumulation patterns

• Evaluate the effects of genome editing on target protein expression

• Investigate relationships between zein structure, protein body formation, and nutritional quality

This research could contribute to developing maize varieties with improved protein quality for human and animal nutrition.

What role might AZS22-14 Antibody play in understanding structure-function relationships of storage proteins?

For structure-function research:

• Use immunolocalization to study the spatial organization of zeins within protein bodies

• Correlate protein modifications with functional properties and processing quality

• Study interactions between different zein classes and their impact on protein body assembly

• Investigate relationships between zein structure and functionality in food systems

• Examine how specific amino acid composition influences protein stability and digestibility

This research would advance our understanding of how protein structure affects functional properties in both plants and food systems.