IRX6 Antibody

What is IRX6 and why is it studied?

IRX6 (Iroquois Homeobox 6) is a member of the Iroquois homeobox gene family. These genes play multiple critical roles during pattern formation of vertebrate embryos during development . As a transcription factor containing a homeodomain, IRX6 is involved in regulating gene expression during embryonic development, making it an important target for developmental biology research and potential disease associations. Understanding its expression patterns can provide insights into normal development and pathological conditions .

What types of IRX6 antibodies are commercially available?

There are multiple types of IRX6 antibodies available, targeting different regions of the protein. These include antibodies targeting the N-terminal region, C-terminal region, and various specific amino acid sequences (e.g., AA 337-446, AA 289-389, AA 260-288) . Both polyclonal and monoclonal antibodies are available, with polyclonal being more common . Most are produced in rabbit hosts and are typically unconjugated, though specific configurations may vary depending on research requirements .

What are the common applications for IRX6 antibodies?

IRX6 antibodies are primarily used in Western Blotting (WB), Immunohistochemistry (IHC), and Enzyme-Linked Immunosorbent Assay (ELISA) . Western Blotting applications typically use dilutions ranging from 1:500-1:1000, while Immunohistochemistry applications usually require dilutions of 1:50-1:500 . Some specialized antibodies may also be applicable for RNA interference (RNAi) studies . These techniques allow researchers to detect, quantify, and localize IRX6 protein in various experimental contexts.

How should researchers optimize Western Blotting protocols for IRX6 detection?

For Western Blotting applications using IRX6 antibodies, researchers should use concentrations between 0.2-1 μg/mL or dilutions of 1:500-1:1000 . The optimal conditions should be determined experimentally for each specific application and sample type. Positive controls such as mouse heart tissue have been validated for certain IRX6 antibodies . When optimizing Western Blotting protocols, consider using blocking peptides (such as catalog no. 33R-8379) as negative controls to confirm specificity . The observed molecular weight for IRX6 is approximately 48 kDa, which matches its calculated molecular weight based on its 446 amino acid sequence .

What are the critical considerations for Immunohistochemistry with IRX6 antibodies?

For Immunohistochemistry applications, researchers should use dilutions between 1:50-1:500 . Antigen retrieval is crucial for optimal results, with recommended methods including TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 . Validated positive control tissues include human colon cancer tissue and mouse heart tissue . Researchers should titrate the antibody for each testing system to obtain optimal results, as the effectiveness may be sample-dependent. Comprehensive validation data galleries are available from some manufacturers to guide optimization efforts .

How should IRX6 antibodies be stored and handled for maximum effectiveness?

IRX6 antibodies should typically be stored at -20°C, where they remain stable for one year after shipment . Most preparations contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 to maintain stability . Aliquoting is generally unnecessary for -20°C storage, but some preparations (particularly 20μL sizes) may contain 0.1% BSA as an additional stabilizer . When shipping or transporting antibodies, they should be kept on wet ice to maintain their integrity . Always avoid repeated freeze-thaw cycles as this can compromise antibody activity.

What species reactivity is observed with different IRX6 antibodies?

IRX6 antibodies show varying species reactivity depending on the specific product and epitope targeted. Common reactivity profiles include:

• Antibodies reacting with human, rat, and mouse samples

• Antibodies with broader reactivity profiles extending to cow, horse, dog, guinea pig, and pig

• Some antibodies showing reactivity with primates (monkey)

• Antibodies specifically designed for human-only applications

When selecting an antibody, researchers should verify the cross-reactivity profile against their target species to ensure compatibility with their experimental model system .

How does epitope selection impact IRX6 antibody performance?

The epitope targeted by IRX6 antibodies significantly impacts their performance and specificity. Antibodies are available targeting various regions:

• N-Terminal antibodies offer broad species cross-reactivity

• C-Terminal antibodies provide alternative epitope recognition

• Specific amino acid sequence-targeted antibodies (e.g., AA 337-446, AA 289-389) provide options for recognizing particular domains

• Full-length antibodies (AA 1-446) may offer broader epitope recognition

Researchers should select antibodies targeting regions relevant to their experimental question, considering factors such as protein structure, post-translational modifications, protein-protein interactions, and accessibility of the epitope under experimental conditions .

How can IRX6 antibodies be used to study developmental biology?

IRX6, as a member of the Iroquois homeobox gene family, plays crucial roles in pattern formation during vertebrate embryonic development . Researchers can use IRX6 antibodies to map expression patterns across developmental stages and tissue types using immunohistochemistry or immunofluorescence. The Human Protein Atlas project provides valuable data on IRX6 expression patterns in various tissues and at subcellular levels . When designing developmental studies, researchers should consider tissue-specific expression patterns, temporal regulation, and potential interactions with other developmental regulators. Comparative studies across species can provide evolutionary insights into conserved developmental mechanisms.

What controls should be incorporated in IRX6 antibody-based experiments?

Proper experimental controls are essential for reliable results with IRX6 antibodies. Positive controls should include tissues known to express IRX6, such as mouse heart tissue or human colon cancer tissue . Negative controls should include tissues known not to express IRX6 or experiments using blocking peptides specific to the antibody (e.g., catalog no. 33R-8379) . Isotype controls using non-specific IgG from the same host species (typically rabbit) at equivalent concentrations can help distinguish specific from non-specific binding. For quantitative applications, standard curves using recombinant IRX6 protein can improve accuracy and reproducibility.

What are the considerations for multiplexing IRX6 antibodies with other markers?

When designing multiplexed immunoassays including IRX6, researchers should consider several factors. First, select antibodies raised in different host species to avoid cross-reactivity between secondary antibodies. If using multiple rabbit antibodies, consider directly conjugated primary antibodies or sequential immunostaining protocols with intermediate blocking steps. Fluorophore selection should account for spectral overlap and tissue autofluorescence. Titration of each antibody in the multiplex panel is crucial, as optimal concentrations may differ from single-staining protocols. Comprehensive controls, including single-stain controls and fluorescence-minus-one controls, are essential for accurate interpretation of multiplexed data.

What are common troubleshooting strategies for Western Blotting with IRX6 antibodies?

When encountering challenges with Western Blotting using IRX6 antibodies, several troubleshooting approaches can be employed. If signal is weak or absent, consider increasing antibody concentration, extending incubation time, or using enhanced detection systems. The recommended concentration range of 0.2-1 μg/mL should be optimized for specific sample types . For high background, optimize blocking conditions, increase washing steps, or reduce antibody concentration. If multiple bands appear, evaluate sample preparation methods, consider using reducing vs. non-reducing conditions, and verify with blocking peptides to confirm specificity . For inconsistent results across experiments, standardize protein loading, transfer conditions, and consider using housekeeping protein controls.

How can researchers address challenges in Immunohistochemistry with IRX6 antibodies?

For optimal Immunohistochemistry results with IRX6 antibodies, antigen retrieval is critical, with recommended buffers including TE buffer (pH 9.0) or citrate buffer (pH 6.0) . If staining is weak, increase antibody concentration within the recommended range (1:50-1:500), extend incubation time, or optimize antigen retrieval conditions . For background issues, increase blocking time, optimize antibody dilution, or use additional blocking agents. False positives can be addressed by comparing to known positive tissues (mouse heart, human colon cancer) and using appropriate negative controls . Tissue-specific optimization may be necessary, as the effectiveness of the antibody can be sample-dependent .

What considerations should be made when validating a new IRX6 antibody lot?

When validating a new lot of IRX6 antibody, researchers should compare performance against previous lots using consistent protocols. This validation should include testing on known positive controls (mouse heart tissue, human colon cancer tissue) , verification of expected band size (~48 kDa) in Western Blotting , comparison of staining patterns in Immunohistochemistry, and assessment of background levels. Lot-to-lot variability may require adjustment of working concentrations or incubation times. Documentation of validation results, including images and quantitative measurements, allows for long-term monitoring of antibody performance across projects and facilitates troubleshooting if performance issues arise.