EIF2AK3 Antibody, Biotin conjugated

What are the specifications of biotin-conjugated EIF2AK3 antibodies?

Biotin-conjugated EIF2AK3 antibodies are specifically designed for enhanced detection in assays requiring signal amplification, particularly ELISA applications . These antibodies typically use rabbit-derived IgG as the host isotype and are supplied in liquid form . The antibodies target the human EIF2AK3 protein (UniProt ID: Q9NZJ5) and are commonly generated using recombinant human EIF2AK3 protein fragments as immunogens. For example, the Assay Genie product uses recombinant human EIF2AK3 protein fragment spanning amino acids 115-330 as the immunogen . Storage recommendations include maintaining the antibody at -20°C in buffer solutions containing preservatives such as 0.03% Proclin 300 and stabilizers like 50% glycerol in PBS at pH 7.4 .

What applications are most suitable for biotin-conjugated EIF2AK3 antibodies?

Biotin-conjugated EIF2AK3 antibodies are primarily optimized for ELISA applications . In sandwich ELISA formats, these conjugated antibodies serve as detection antibodies that bind to the target EIF2AK3 protein already captured by pre-coated primary antibodies . This allows for the subsequent binding of HRP-Streptavidin, which interacts with the biotin conjugate to generate a colorimetric signal when appropriate substrates (such as TMB) are added . While unconjugated EIF2AK3 antibodies have demonstrated efficacy in Western blot, immunohistochemistry, immunofluorescence, and co-immunoprecipitation applications, biotin-conjugated versions are specially optimized for the signal amplification requirements of ELISA protocols .

How should I optimize ELISA protocols using biotin-conjugated EIF2AK3 antibodies?

For optimal ELISA performance using biotin-conjugated EIF2AK3 antibodies, follow this detailed methodology based on established sandwich ELISA principles. Begin with a 96-well plate pre-coated with an anti-EIF2AK3 capture antibody . After blocking, add standards and samples to the wells according to your experimental design. The biotin-conjugated anti-EIF2AK3 antibody functions as the detection antibody in this system . Following sample incubation and washing to remove unbound materials, add the biotinylated detection antibody which binds to EIF2AK3 already captured on the plate .

After thorough washing to remove excess detection antibody, add HRP-Streptavidin which binds with high affinity to the biotin conjugate . Following another washing step, add TMB substrate which is catalyzed by HRP to produce a blue color product that turns yellow after adding the stop solution . The optical density should be read at 450nm, with the concentration of EIF2AK3 in your samples calculated against a standard curve . For optimal results, it is recommended that you titrate the biotin-conjugated antibody in your specific testing system rather than relying solely on recommended dilutions .

Why is there a discrepancy between calculated and observed molecular weights of EIF2AK3?

The EIF2AK3/PERK protein has a calculated molecular weight of 125 kDa based on its 1116 amino acid sequence, but is frequently observed at approximately 140 kDa in experimental systems . This 15 kDa difference is attributed to post-translational modifications of the protein, particularly phosphorylation events that occur during activation of the kinase . In some cases, EIF2AK3 may even be detected at 170 kDa due to extensive modifications . These modifications are functionally significant as they reflect the activation state of the kinase in response to cellular stress conditions.

When validating EIF2AK3 antibody specificity, researchers should expect bands at approximately 140 kDa rather than at the calculated 125 kDa position. The observed molecular weight can vary depending on the cell type, stress conditions, and activation state of the kinase . This information is particularly important when interpreting Western blot results and in distinguishing between inactive and active forms of the protein in experimental systems studying the unfolded protein response or other stress pathways.

What controls are essential when using EIF2AK3 antibodies in research applications?

When designing experiments with EIF2AK3 antibodies, including the biotin-conjugated format, several controls are critical for ensuring valid and interpretable results. Positive cellular controls should include cell lines known to express EIF2AK3, such as HEK-293, HepG2, and MCF-7 cells for Western blotting, and HeLa cells for flow cytometry applications . For immunohistochemistry, human pancreatic cancer tissue has been validated as a positive control material .

For negative controls, consider using EIF2AK3 knockout or knockdown cells, as evidenced by the 4 published studies cited in the antibody validation data . When validating antibody specificity, blocking peptides corresponding to the immunogen sequence can help confirm binding specificity. For ELISA applications specifically using biotin-conjugated antibodies, include controls without the primary antibody, without the biotinylated detection antibody, and without the HRP-Streptavidin to identify sources of non-specific signal . Additionally, incorporating a standard curve using recombinant EIF2AK3 protein allows for quantitative assessment and ensures the assay is functioning within its linear range .

What are the recommended dilutions for EIF2AK3 antibodies across different applications?

Optimal dilutions for EIF2AK3 antibodies vary significantly based on the specific application and experimental system. Based on extensive validation data, the following dilution ranges are recommended:

For biotin-conjugated antibodies specifically used in ELISA applications, dilution optimization is particularly important as the signal amplification properties of the biotin-streptavidin system can lead to high background if not properly titrated . It is strongly recommended to perform a titration series in each specific experimental system rather than relying solely on the recommended ranges .

How can I optimize antigen retrieval for EIF2AK3 detection in tissue sections?

For optimal EIF2AK3 detection in fixed tissue sections, antigen retrieval methodology is critical. Based on validation data with human pancreatic cancer tissue, TE buffer at pH 9.0 is the primary recommended buffer for antigen retrieval when using anti-EIF2AK3 antibodies for immunohistochemistry . As an alternative, citrate buffer at pH 6.0 may also be used, though potentially with reduced efficacy .

The choice between these two methodologies depends on your specific tissue type and fixation protocol. For formalin-fixed, paraffin-embedded (FFPE) tissues with extensive crosslinking, the alkaline TE buffer (pH 9.0) typically provides superior epitope unmasking. For tissues with milder fixation or when working with frozen sections, the milder citrate buffer approach may be sufficient. When developing a new protocol for previously untested tissue types, it is advisable to perform a side-by-side comparison of both antigen retrieval methods to determine which provides optimal signal-to-noise ratio for your specific experimental conditions .

How can I quantitatively assess EIF2AK3 activation in stress response studies?

To quantitatively assess EIF2AK3 activation in experimental systems studying stress responses, researchers should employ a multi-parameter approach. Since EIF2AK3 activation occurs through auto-phosphorylation and subsequent phosphorylation of eIF2α, both events should be monitored. Western blotting using phospho-specific antibodies against activated EIF2AK3 and its downstream target phospho-eIF2α (Ser51) provides direct evidence of the activation pathway . The ratio of phosphorylated to total protein for both EIF2AK3 and eIF2α offers a quantitative measure of pathway activation.

Flow cytometry using intracellular staining with the appropriate antibodies (such as CL488-24390) allows for single-cell resolution analysis of EIF2AK3 activation in heterogeneous populations . For high-throughput analysis, ELISA-based assays using biotin-conjugated antibodies can quantify EIF2AK3 levels across multiple samples . Additionally, monitoring downstream effects such as ATF4 upregulation and subsequent target gene expression provides functional validation of the EIF2AK3 signaling cascade . Researchers should incorporate appropriate positive controls such as tunicamycin or thapsigargin treatment to induce ER stress and negative controls using EIF2AK3 inhibitors or knockdown systems to validate the specificity of observed activation patterns.

How do I troubleshoot weak or non-specific signals when using EIF2AK3 antibodies?

When encountering weak or non-specific signals with EIF2AK3 antibodies, including biotin-conjugated formats, consider these application-specific troubleshooting approaches:

For weak signals in Western blotting, try increasing the antibody concentration within the recommended range (1:500-1:2000), extending primary antibody incubation time (overnight at 4°C), or using enhanced chemiluminescence substrates . For ELISA applications with biotin-conjugated antibodies, optimize each component of the detection system including capture antibody concentration, biotinylated detection antibody dilution, and HRP-Streptavidin concentration .

To address non-specific binding in immunohistochemistry, optimize blocking conditions using 5-10% normal serum from the same species as the secondary antibody, and consider antigen retrieval optimization between TE buffer pH 9.0 and citrate buffer pH 6.0 . For flow cytometry applications, increase washing steps and optimize fixation/permeabilization protocols for intracellular staining .

If multiple bands appear in Western blotting, this could reflect EIF2AK3 isoforms, degradation products, or post-translational modifications. The observed molecular weight of EIF2AK3 (140 kDa) differs from the calculated weight (125 kDa) due to modifications, which should be considered when interpreting results . Validation with positive controls (HEK-293, HepG2, or MCF-7 cells) and negative controls (knockdown/knockout samples) can help confirm antibody specificity .

How can I integrate EIF2AK3 detection into multiplex assays studying the unfolded protein response?

For comprehensive analysis of the unfolded protein response (UPR) pathway, integrating EIF2AK3/PERK detection into multiplex assays provides valuable insights into the coordinated stress response. For immunofluorescence multiplex assays, combine fluorescently-labeled EIF2AK3 antibodies like CL488-24390 (excitation/emission: 493/522 nm) with antibodies against other UPR pathway components such as IRE1α and ATF6, using fluorophores with distinct spectral properties to avoid overlap .

In flow cytometry-based multiplex analysis, utilize the biotin-conjugated EIF2AK3 antibody with streptavidin coupled to a fluorophore that complements your panel design, while carefully titrating each antibody to minimize compensation requirements . For protein array or bead-based multiplex systems, the biotin-conjugated EIF2AK3 antibody can be incorporated into panels detecting multiple UPR components simultaneously .

When designing these multiplex approaches, key considerations include antibody compatibility (avoiding host species cross-reactivity), optimization of fixation and permeabilization protocols to preserve epitopes for all targets, and implementation of appropriate compensation controls for spectral overlap in fluorescence-based systems. Validation should include single-stain controls for each antibody and biological controls with known activation states of the UPR pathway components .

How can EIF2AK3 antibodies contribute to studies of disease mechanisms?

EIF2AK3/PERK antibodies, including biotin-conjugated formats, have significant applications in disease mechanism research, particularly in conditions involving endoplasmic reticulum stress and the unfolded protein response. Defects in EIF2AK3 are causative in Wolcott-Rallison syndrome (WRS), making these antibodies valuable tools for investigating this rare genetic disorder . In cancer research, EIF2AK3 antibodies have been validated for detecting expression in pancreatic cancer tissue, supporting studies into how UPR modulation affects tumor cell survival under metabolic stress conditions .

Neurodegenerative diseases such as Alzheimer's and Parkinson's disease involve protein misfolding and ER stress, making EIF2AK3 detection critical for understanding disease progression and potential therapeutic interventions. In diabetes research, EIF2AK3's role in pancreatic β-cell function and survival under ER stress conditions has positioned it as an important biomarker for islet health and potential therapeutic target .

For researchers investigating these disease mechanisms, combining EIF2AK3 antibody detection methods with functional readouts of pathway activation (such as downstream target expression) provides a comprehensive understanding of how alterations in this stress response pathway contribute to pathogenesis. The availability of multiple detection formats, including biotin-conjugated antibodies for sensitive ELISA-based screening, enables researchers to select the optimal approach for their specific disease model and experimental questions .

What recent advances have improved EIF2AK3 antibody specificity and applications?

Recent technological advancements have significantly enhanced both the specificity and application range of EIF2AK3 antibodies. The development of purification methods using antigen affinity techniques has improved antibody specificity compared to traditional methods . Modern immunogens utilizing recombinant protein fragments, such as the 115-330 amino acid region of human EIF2AK3, have enabled the generation of antibodies with superior epitope recognition and reduced cross-reactivity .

Conjugation chemistry advancements have expanded the utility of EIF2AK3 antibodies, with options now including biotin conjugates for ELISA applications and fluorescent conjugates such as CoraLite® Plus 488 for direct detection in flow cytometry and imaging . These conjugated formats eliminate the need for secondary detection reagents, reducing background and improving signal-to-noise ratios in complex experimental systems.

Validation methodology has also evolved, with antibodies now being tested against knockdown/knockout controls to confirm specificity, as evidenced by the 4 published studies cited in antibody validation data . Additionally, cross-species reactivity testing has identified antibodies capable of recognizing EIF2AK3 across human, porcine, and bovine samples, facilitating comparative studies across model systems . These combined advances have enhanced the reliability and versatility of EIF2AK3 antibodies as research tools for investigating stress response pathways in both normal physiological and pathological contexts.