ANKRD2 Antibody

What is the optimal application range for ANKRD2 antibody?

ANKRD2 antibody can be effectively used across multiple experimental applications with specific recommended dilutions:

It is important to note that these dilutions may need optimization for specific experimental systems to obtain optimal results, and sample-dependent variations have been observed . Most published research applications have utilized this antibody for Western blot analysis, with several supporting publications documenting its effectiveness .

What tissues show positive reactivity with ANKRD2 antibody?

ANKRD2 antibody shows positive reactivity in multiple tissue types, with strongest detection in muscle tissues. Western blot analysis has confirmed positive detection in mouse and human skeletal muscle tissue . Immunohistochemistry has demonstrated positive detection in human kidney, lung, skeletal muscle, testis, brain, and skin tissues . For optimal IHC results, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may also be used as an alternative . The differential expression pattern of ANKRD2 protein correlates with the composition of slow muscle fibers in various tissues .

How should ANKRD2 antibody be stored for optimal stability?

The ANKRD2 antibody should be stored at -20°C, where it remains stable for one year after shipment . The storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is unnecessary for storage at -20°C . For smaller 20μl size formats, the product contains 0.1% BSA, which should be taken into consideration when designing experiments that may be sensitive to bovine serum albumin .

What is the expected molecular weight of ANKRD2 when performing Western blot?

Although the calculated molecular weight of ANKRD2 is 37 kDa (aa) and 2 kDa, Western blot analysis typically detects the protein at approximately 42 kDa . This discrepancy between calculated and observed molecular weight is important to note when interpreting experimental results. Interestingly, the antibody also recognizes trace amounts of a 55-kDa protein in skeletal muscles . This 55-kDa band might represent a precursor form of ANKRD2 or result from translation initiation from an upstream in-frame ATG site . While the main 37-kDa ANKRD2 protein is undetectable in cardiac muscle, the 55-kDa protein is highly expressed in this tissue .

How can ANKRD2 antibody be utilized to study differential expression in fast versus slow muscle fibers?

ANKRD2 antibody is an excellent tool for studying muscle fiber type differences due to the protein's differential expression pattern. Research shows that slow postural soleus muscle, which expresses high levels of type 1 myosin heavy chain isoform, contains significantly more ANKRD2 protein compared to fast tibialis anterior (TA) and extensor digitorum longus (EDL) muscles . When designing experiments to study this distribution, researchers should include appropriate muscle type controls and potentially co-stain with fiber-type specific markers.

For denervation studies, ANKRD2 antibody can track phenotypic changes, as ANKRD2 is dramatically downregulated in denervated slow-twitch soleus muscle, correlating with the slow-to-fast fiber transition . In experimental protocols, Western blot analysis can detect this downregulation as early as 1 week post-denervation, with ANKRD2 becoming barely detectable after 2-4 weeks . For extremely low expression levels, more sensitive proteomics approaches like Ciphergen IDM bead-based methods may be necessary to detect residual ANKRD2 protein .

What experimental considerations are important when studying ANKRD2 in mechanotransduction pathways?

When designing experiments to study ANKRD2's role in mechanotransduction, researchers should consider that passive stretch of skeletal muscle leads to significant upregulation of both ANKRD2 mRNA and protein levels (p > 0.001) in fast tibialis anterior muscle after 4-7 days of stretch . This upregulation appears to be associated with the stretch-induced expression of slow muscle phenotype rather than the hypertrophic response .

To effectively study this phenomenon, experimental designs should:

• Include appropriate time points (4-7 days of stretch shows significant changes)

• Compare stretched versus non-stretched control muscles

• Consider parallel analysis of slow muscle phenotype markers

• Potentially examine transcription factors known to interact with ANKRD2, such as YB-1, p53, and promyelocytic leukemia protein

The antibody can be used in combination with transcript analysis to provide comprehensive insights into ANKRD2's role in mechanical signal transduction pathways in muscle tissue.

How can ANKRD2 antibody be employed in cancer research, particularly osteosarcoma studies?

Recent research has implicated ANKRD2 in osteosarcoma progression, opening new applications for ANKRD2 antibody in cancer research . When designing experiments to study ANKRD2's role in osteosarcoma, researchers should consider both gain-of-function and loss-of-function approaches.

Exogenous expression of ANKRD2 has been shown to influence cellular growth, migration, and clonogenicity in a cell line-dependent manner . Furthermore, ANKRD2 expression enhances 3D spheroid formation in multiple cellular models and increases matrix metalloproteinase (MMP) activity . Conversely, downregulation of ANKRD2 reduces proliferation and clonogenic potential .

For these studies, ANKRD2 antibody can be used in Western blot analysis to confirm expression or knockdown, in immunofluorescence to examine subcellular localization, and potentially in co-immunoprecipitation experiments to identify interacting partners in cancer cells. When designing experiments, researchers should consider:

• Using multiple osteosarcoma cell lines due to cell line-dependent effects

• Including 3D culture models alongside traditional 2D cultures

• Examining both proliferation and migration/invasion endpoints

• Analyzing MMP activity and expression (particularly MMP2)

What approaches can be used to study the different isoforms of ANKRD2 detected by the antibody?

The ANKRD2 antibody detects both the primary 37-kDa ANKRD2 protein and a 55-kDa protein that may represent an alternative isoform . To effectively study these different isoforms, researchers can implement several approaches:

• Differential tissue analysis: The 55-kDa protein is highly expressed in cardiac muscle where the 37-kDa form is undetectable, making cardiac tissue an excellent control for studying the larger isoform .

• Denervation models: Interestingly, the 55-kDa protein shows an opposite expression pattern compared to the 37-kDa protein in denervated muscle, providing a useful experimental model to distinguish between the isoforms .

• Molecular analysis: PCR with primers designed to detect potential alternative splicing or alternative translation start sites can complement protein analysis. For PCR, conditions similar to those described in the literature can be used: 1 cycle at 95°C for 3 min; 35 cycles for ANKRD2 or 30 cycles for GAPDH at 95°C for 30s, 60°C for 35s, and 72°C for 30s; followed by 1 cycle at 72°C for 10 min .

• Subcellular fractionation: As ANKRD2 has been reported to have both cytoplasmic and nuclear localization sequences, fractionation followed by Western blot can help determine if the different isoforms have distinct subcellular distributions.

How can ANKRD2 antibody be used to study post-translational modifications of the protein?

ANKRD2 undergoes phosphorylation by Akt2 on serine 99 (S99) upon exposure to oxidative stress, which has significant functional consequences including negative effects on muscle differentiation . When designing experiments to study these modifications:

• Phospho-specific detection: While standard ANKRD2 antibody detects total ANKRD2 protein, phospho-specific antibodies would be needed to directly detect the S99 phosphorylation state. In the absence of commercial phospho-specific antibodies, researchers might:

  • Use phospho-protein enrichment followed by ANKRD2 detection

  • Employ phosphatase treatments as controls to confirm phosphorylation status

  • Use mobility shift analysis, as phosphorylation may alter protein migration

• Induction protocols: Oxidative stress can be induced experimentally to trigger ANKRD2 phosphorylation, for example using H₂O₂ treatment .

• Co-detection with signaling proteins: Co-immunoprecipitation or co-staining with anti-Akt and anti-phospho-Akt antibodies can help establish the relationship between ANKRD2 and its upstream kinase .

• Functional assays: The impact of phosphorylation can be studied by comparing wild-type ANKRD2 with phospho-mimetic or phospho-null mutants in functional assays, which has previously revealed that unphosphorylatable forms decrease differentiation rate in transfected cells .

What are the key considerations when validating new applications of ANKRD2 antibody?

When validating ANKRD2 antibody for new applications, researchers should consider several important factors:

• Appropriate controls: Include both positive controls (tissues known to express ANKRD2, such as skeletal muscle) and negative controls (tissues with minimal expression or ANKRD2 knockdown samples) .

• Dilution optimization: While recommended dilutions provide a starting point, each experimental system may require titration to achieve optimal signal-to-noise ratios .

• Isoform awareness: Be conscious of the potential detection of multiple isoforms (37-kDa and 55-kDa proteins) when interpreting results, particularly in cardiac tissue where the expression pattern differs significantly from skeletal muscle .

• Cross-validation: When possible, validate findings using complementary techniques such as RT-PCR for transcript levels alongside protein detection methods .

• Species considerations: While the antibody shows reactivity with human, mouse, and rat samples, expression patterns and molecular weights may vary between species, requiring careful interpretation .