TSC22D1 Antibody

What is the molecular structure of TSC22D1 and how does it affect antibody selection?

TSC22D1 exists in multiple isoforms with different molecular weights due to alternative start codon usage. The primary isoforms include TSC22D1.1 (long variant) and TSC22D1.2 (short variant). The short variant (TSC22D1.2) produces three distinct protein products with apparent molecular weights of approximately 18 kDa, 17 kDa, and 14 kDa due to differential start codon usage from the same transcript . When selecting antibodies, researchers must consider which epitopes are present in their protein of interest, as some antibodies detect only specific isoforms.

The longest encoded protein of TSC22D1.2 contains 144 amino acids with a predicted size of 18 kDa, while alternative translation initiation sites produce proteins of 134 aa and 86 aa (17 kDa and 14 kDa, respectively) . Researchers should verify antibody documentation to determine which protein variants will be detected in their experimental system.

Which tissues and cell types express TSC22D1 protein?

TSC22D1 protein demonstrates widespread but tissue-specific expression patterns. Based on immunohistochemistry studies, TSC22D1 is detected in:

Subcellular localization studies using immunofluorescence in HEK293 cells reveal that endogenous TSC22D1 primarily localizes to the mitochondria and nucleus, with TSC22D1-2 found in both compartments while TSC22D1-1 is predominantly nuclear . This differential localization may influence experimental design when working with specific cellular fractions.

What are the recommended applications for TSC22D1 antibodies?

TSC22D1 antibodies have been validated for multiple applications with specific dilution recommendations:

For optimal results, each antibody should be titrated in the specific experimental system, as sensitivity may vary between tissue types and preparation methods .

How should I optimize Western blot protocols for detecting TSC22D1 isoforms?

Western blot detection of TSC22D1 requires careful consideration of sample preparation and gel resolution to distinguish between isoforms:

• Sample preparation: For optimal detection of all TSC22D1 isoforms, use protease inhibitors during extraction, as the smaller isoforms (14 kDa and 17 kDa) are not degradation products but result from alternative start codon usage .

• Gel selection: Use 12-15% polyacrylamide gels to achieve sufficient resolution between the closely migrating bands (18 kDa, 17 kDa, and 14 kDa).

• Antibody selection: Verify that your antibody can detect multiple isoforms if needed. For example, antibody 10214-1-Ig has been validated to detect TSC22D1 at its observed molecular weight of 16 kDa .

• Controls: Include positive controls such as human or rat brain tissue lysates, which consistently show strong TSC22D1 expression . For knockout validation, cells treated with TSC22D1-targeting shRNAs (sh-TSC22(1) and sh-TSC22(2)) can serve as negative controls .

• Blotting conditions: Dilute primary antibody between 1:500-1:1000 in blocking buffer. Incubation at 4°C overnight typically yields better results than shorter incubations at room temperature .

What are the critical considerations for immunohistochemical detection of TSC22D1?

Successful IHC detection of TSC22D1 requires optimization of several parameters:

• Antigen retrieval: For formalin-fixed paraffin-embedded tissues, use TE buffer at pH 9.0 for optimal antigen retrieval. Alternatively, citrate buffer at pH 6.0 may be used, though potentially with reduced effectiveness .

• Antibody dilution: Start with a dilution range of 1:20-1:200 and optimize based on signal-to-background ratio .

• Positive control tissues: Include human brain tissue or human testis tissue as positive controls, which consistently show strong TSC22D1 expression .

• Visualization system: Use either HRP-DAB or fluorescent secondary antibodies depending on the desired analysis method and sensitivity requirements.

• Counterstaining: Hematoxylin works well as a counterstain for DAB detection systems, allowing visualization of tissue architecture while maintaining TSC22D1 signal integrity.

Researchers have successfully detected TSC22D1 in human brain and testis tissues using a 1:50 dilution with commercially available antibodies such as Catalog No:116423 .

How can I investigate TSC22D1 protein-protein interactions using immunoprecipitation?

TSC22D1 interacts with multiple proteins that regulate its function in cellular processes. To study these interactions:

• Cell/tissue selection: Rat brain tissue and HEK293 cells have been successfully used for TSC22D1 IP studies . For brain tissue, use 3-4 mg of total protein lysate with 0.5-4.0 μg of antibody .

• IP strategy: Two approaches have been validated:

  • In vitro GST pull-down assay using GST-TSC22 fusion proteins

  • In vivo Flag-tagged TSC22D1 IP from transfected cells

• Binding partner detection: After IP, analyze samples by mass spectrometry or Western blot with antibodies against suspected binding partners. Confirmed binding partners include:

• Control IPs: Include negative controls (non-specific IgG) and isoform-specific controls (comparing TSC22D1.2 vs. TSC22(86) binding partners) .

Research has shown that the binding proteins identified by in vitro pull-down methods don't always match those found in in vivo binding assays, likely due to differences in protein conformation when using GST (26 kDa) versus Flag (1 kDa) tags .

What approaches can distinguish the roles of different TSC22D1 isoforms in oncogene-induced senescence?

TSC22D1 isoforms play antagonistic roles in BRAF E600-induced senescence. To investigate these functions:

• Isoform-specific expression analysis: Use RT-qPCR with primers specific to TSC22D1.1 and TSC22D1.2 transcripts. In senescent cells, TSC22D1.2 mRNA is upregulated >100-fold while TSC22D1.1 remains unchanged .

• Protein isoform analysis: Western blotting can detect the three protein variants of TSC22D1.2 (18 kDa, 17 kDa, and 14 kDa) in senescent cells. The larger TSC22D1.1 protein is suppressed by proteasomal degradation during senescence .

• Functional studies: Use shRNA-mediated knockdown targeting either shared regions (affecting all isoforms) or isoform-specific regions. Two validated shRNAs (sh-TSC22(1) and sh-TSC22(2)) show different efficacies in depleting TSC22D1 .

• Overexpression studies: Express individual isoforms (TSC22D1.1 or TSC22D1.2) in cells using constructs with optimized Kozak sequences to control which protein variant is produced .

• Senescence markers: Assess the impact on senescence using:

  • SA β-galactosidase activity

  • Cell morphology changes (spindle-shaped morphology)

  • Expression of senescence markers (p15INK4B)

  • Inflammatory factors (IL6, IL8, IL1β)

Research has shown that depleting the short TSC22D1.2 form or overexpressing the large TSC22D1.1 variant results in abrogation of oncogene-induced senescence, demonstrating their antagonistic functions .

Why might I detect multiple bands when using TSC22D1 antibodies in Western blot?

Multiple bands in TSC22D1 Western blots are often biologically relevant rather than artifacts. Consider these explanations:

• Alternative translation initiation: The TSC22D1.2 transcript produces three protein variants (18 kDa, 17 kDa, and 14 kDa) due to alternative translation initiation at different ATG codons. These represent distinct biological entities, not degradation products .

• Different isoforms: TSC22D1.1 (longer isoform) and TSC22D1.2 (shorter isoform) are produced from alternative splicing and will appear as distinct bands .

• Post-translational modifications: Phosphorylation or other modifications may cause shifts in apparent molecular weight.

• Tissue-specific expression: Different tissues may express different ratios of TSC22D1 isoforms. For example, in senescent cells, the shorter TSC22D1.2 variants (14 kDa and 17 kDa) are predominant while the 18 kDa form is weakly expressed .

To distinguish between these possibilities:

• Use positive controls with known isoform expression patterns

• Include isoform-specific recombinant protein standards

• Perform proteasome inhibition experiments (proteasome inhibition increases rather than decreases the levels of the smallest TSC22D1.2 proteins, confirming they are not degradation products)

What are the optimal storage conditions for TSC22D1 antibodies to maintain reactivity?

Proper storage is critical for maintaining antibody performance across experiments:

• Short-term storage: For immediate use within two weeks, store at 4°C .

• Long-term storage: Divide into small aliquots (≥20 μl) and store at -20°C or -80°C to avoid repeated freeze-thaw cycles . Most commercial TSC22D1 antibodies are stable for one year after shipment when stored properly .

• Storage buffer: Most commercial TSC22D1 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . This formulation helps maintain stability during freezing.

• Handling precautions:

  • Avoid repeated freeze-thaw cycles as they can decrease antibody activity

  • For concentrated products, adding an equal volume of glycerol as a cryoprotectant prior to freezing is recommended

  • Allow antibodies to warm to room temperature before opening to prevent condensation

Manufacturers typically provide specific storage recommendations for individual antibody products, which may vary slightly. For example, product 10214-1-Ig specifies that aliquoting is unnecessary for -20°C storage, while 20μl sizes contain 0.1% BSA for additional stability .

How can TSC22D1 antibodies be used to investigate its role in cancer biology?

TSC22D1 has been implicated in tumor suppression, with specific roles in different cancer types. Research approaches include:

• Expression analysis in cancer tissues: IHC studies have validated TSC22D1 antibodies for detection in human brain tissues and human gliomas . Compare expression levels between normal and malignant tissues using standardized IHC protocols.

• Analysis of isoform balance: The antagonistic roles of TSC22D1 isoforms in cancer progression can be investigated using isoform-specific detection. In BRAF E600-driven neoplasia, the balance between short and long TSC22D1 variants appears critical .

• Pathway interaction studies: TSC22D1 interacts with p15INK4B and inflammatory factors in the context of oncogene-induced senescence . Co-IP studies can reveal cancer-specific protein interactions.

• Functional studies in cancer cell lines: TSC22D1 antibodies have been validated in A549 lung adenocarcinoma cells for WB and IF/ICC applications . Knockdown studies combined with phenotypic assays can reveal cancer-specific functions.

• Biomarker potential: A single nucleotide polymorphism in the TSC22D1 promoter has been associated with specific diseases, suggesting potential as a biomarker . Antibodies can help correlate protein expression with genetic variants.

Research has shown that TSC22D1 transgenic mice frequently develop B cell lymphoma, indicating its relevance to hematological malignancies . Additionally, TSC22D1 has been shown to increase the susceptibility of salivary gland cancer cells (TYS cells) to chemotherapy and radiotherapy .

What approaches can determine how TSC22D1 subcellular localization changes in response to cellular stress?

TSC22D1 localization is dynamically regulated in response to cellular stress, which can be studied using these approaches:

• Stress induction protocols:

  • DNA damage: Induce with agents like UV radiation or etoposide

  • Cytotoxic stress: Use chemical inducers relevant to the cell type

• Subcellular fractionation: Separate nuclear, cytoplasmic, and mitochondrial fractions before Western blot analysis. Use fraction-specific markers such as:

  • Tom20 for mitochondrial fraction

  • Lamin B1 for nuclear fraction

• Immunofluorescence microscopy: Track TSC22D1 localization in live or fixed cells using validated antibodies at 1:200-1:800 dilution . Co-stain with organelle markers like:

  • Tim50 for mitochondria

  • DAPI for nucleus

• Quantitative analysis: Measure nuclear/cytoplasmic ratios of TSC22D1 staining intensity before and after stress induction.

Research has demonstrated that TSC22D1 is localized in the cytoplasm by its nuclear export signal (NES) under normal conditions but translocates to the nucleus under stress conditions . This translocation appears functionally important, as nuclear TSC22D1 interacts with proteins like histone H1.2 .

Immunofluorescence studies in HEK293 cells revealed that endogenous TSC22D1 appears as dots in the cytoplasm (primarily mitochondrial) with additional nuclear fluorescence . Western blotting of subcellular fractions confirmed that TSC22D1-2 localizes to both mitochondria and nucleus, while TSC22D1-1 is predominantly nuclear .

How can chromatin immunoprecipitation (ChIP) be optimized to study TSC22D1 interactions with DNA?

TSC22D1 functions as a transcription factor, making ChIP a valuable approach to identify its genomic targets:

• Antibody selection: Choose antibodies validated for immunoprecipitation, such as 10214-1-Ig, which has been successfully used for IP of TSC22D1 from rat brain tissue .

• Cross-linking optimization: Since TSC22D1 interacts with histone H1.2 , standard formaldehyde cross-linking (1% for 10 minutes) should be sufficient to capture DNA interactions.

• Chromatin preparation: Sonication conditions should be optimized to generate DNA fragments of 200-500 bp. Test different sonication times in pilot experiments.

• IP controls:

  • Positive control: Use antibodies against known transcription factors

  • Negative control: Use non-specific IgG matching the host species of your TSC22D1 antibody

• Target validation: Verify ChIP enrichment at suspected target genes such as C-type natriuretic peptide, which is regulated by TSC22D1 .

The nuclear localization of TSC22D1, particularly under stress conditions, supports its role as a transcription factor . Its interaction with chromatin-associated proteins like histone H1.2 further suggests direct involvement in transcriptional regulation .

How can multi-omics approaches incorporate TSC22D1 antibodies to understand its regulatory networks?

Integrating TSC22D1 antibody-based techniques with omics approaches can provide comprehensive insights:

• Proteomics integration:

  • IP-MS: Use TSC22D1 antibodies for immunoprecipitation followed by mass spectrometry to identify interaction partners in different cellular contexts

  • Reverse-phase protein arrays: Apply validated antibodies (dilution 1:200-1:800) to detect TSC22D1 across multiple samples

• Genomics correlation:

  • ChIP-seq: Map TSC22D1 binding sites genome-wide and correlate with:

  • RNA-seq: Identify genes differentially expressed upon TSC22D1 manipulation

  • ATAC-seq: Determine if TSC22D1 binding correlates with changes in chromatin accessibility

• Single-cell applications:

  • IF for TSC22D1 combined with single-cell RNA-seq to correlate protein levels with transcriptomic states

  • CyTOF with TSC22D1 antibodies to analyze protein levels across heterogeneous cell populations

• Pathway analyses:

  • Focus on TGF-β signaling pathways, as TSC22D1 was originally identified as TGF-β stimulated clone 22

  • Investigate connections to the C/EBPβ pathway, as TSC22D1 acts as a critical effector of C/EBPβ in senescence contexts

Given TSC22D1's involvement in diverse processes including differentiation, senescence, and tumor suppression, multi-omics approaches can help unravel its context-specific functions and regulatory networks .