TTLL2 Antibody

What is TTLL2 and what is its cellular function?

TTLL2 is a probable tubulin polyglutamylase that generates side chains of glutamate on the gamma-carboxyl group of specific glutamate residues within the C-terminal tail of target proteins. In humans, the canonical protein has a length of 592 amino acid residues and a mass of 67.3 kDa . TTLL2 functions in the process of polyglutamylation, particularly forming polyglutamate side chains on tubulin, which is crucial for microtubule structure and function. The protein likely acts when complexed with other proteins and plays an essential role in the post-translational modification of tubulin proteins . Dysregulation of TTLL2 activity has been linked to neurodevelopmental disorders and cancer, making it a significant target for research in these areas .

How should I choose between polyclonal and monoclonal TTLL2 antibodies?

The choice between polyclonal and monoclonal TTLL2 antibodies depends on your experimental goals. Polyclonal antibodies, which are common for TTLL2 detection, recognize multiple epitopes on the antigen and generally provide stronger signals, making them suitable for applications where sensitivity is paramount. Most commercially available TTLL2 antibodies are polyclonal, raised in rabbits, and have been validated for applications like ELISA, IHC, and Western blot .

Monoclonal antibodies, while less common for TTLL2, offer greater specificity for a single epitope and provide more consistent results between batches. For quantitative studies or when examining specific domains of TTLL2, consider antibodies targeting defined regions, such as those specific to amino acids 401-592 of the human TTLL2 protein . When reproducibility across experiments is critical, monoclonal antibodies may be preferable despite potentially lower sensitivity.

What expression patterns does TTLL2 show across tissues?

TTLL2 is notably expressed in the testis, making this tissue particularly relevant for TTLL2 research . Understanding this tissue-specific expression pattern is important when designing experiments, as it affects the choice of positive controls and experimental models. When working with other tissue types, researchers should validate the presence of TTLL2 before proceeding with complex experiments. Published immunohistochemistry data shows TTLL2 expression in human placenta tissue and pancreatic cancer tissue, indicating its presence in these contexts as well .

What are the optimal conditions for using TTLL2 antibodies in immunohistochemistry?

For immunohistochemistry applications using TTLL2 antibodies, paraffin-embedded tissues have been successfully used with dilutions ranging from 1:20 to 1:200, with 1:100 being commonly reported as effective . The optimal protocol typically involves:

• Deparaffinization and rehydration of tissue sections

• Antigen retrieval (heat-induced epitope retrieval in citrate buffer pH 6.0 is often effective)

• Blocking with appropriate serum (5-10% normal serum from the same species as the secondary antibody)

• Primary antibody incubation with anti-TTLL2 at the recommended dilution (typically 1:100) overnight at 4°C

• Secondary antibody detection and visualization

Successful immunostaining has been documented in human placenta tissue and pancreatic cancer samples . When optimizing your protocol, begin with the manufacturer's recommended dilution and adjust based on signal-to-noise ratio in your specific tissue samples.

How can I optimize Western blot protocols for TTLL2 detection?

For optimal Western blot detection of TTLL2 (67.3 kDa), consider these methodological recommendations:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for efficient extraction of TTLL2 from tissues or cells

• Gel selection: 10% SDS-PAGE gels provide good resolution for the 67.3 kDa TTLL2 protein

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 60 minutes to PVDF membranes

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute TTLL2 antibody according to manufacturer's recommendation (typically 1:1000-1:5000) and incubate overnight at 4°C

• Secondary antibody: Anti-rabbit HRP-conjugated secondary at 1:5000 for 1 hour at room temperature

• Detection: Enhanced chemiluminescence with exposure times optimized based on signal strength

For challenging samples, consider enriching for microtubule-associated proteins through a microtubule co-sedimentation assay prior to Western blotting, which can enhance detection of low-abundance TTLL2.

What controls should be included when using TTLL2 antibodies?

Rigorous experimental design requires appropriate controls when using TTLL2 antibodies:

• Positive control: Include testis tissue or cell extracts where TTLL2 is highly expressed

• Negative control: Use tissues known not to express TTLL2 or include antibody diluent without primary antibody

• Specificity control: Pre-adsorption of the antibody with the immunizing peptide should abolish specific staining

• Loading control: For Western blots, include detection of a housekeeping protein (e.g., β-actin, GAPDH)

• Isotype control: Include a non-specific IgG from the same species as the TTLL2 antibody at the same concentration

When using conjugated TTLL2 antibodies (FITC, HRP, or biotin), include additional controls to account for potential non-specific binding of the conjugate .

How do I design experiments to study TTLL2's role in tubulin polyglutamylation?

Designing experiments to investigate TTLL2's role in tubulin polyglutamylation requires a multi-faceted approach:

• Knockdown/knockout studies: Use siRNA, shRNA, or CRISPR-Cas9 to reduce or eliminate TTLL2 expression, then assess changes in tubulin polyglutamylation using antibodies specific to polyglutamylated tubulin

• Overexpression studies: Express tagged TTLL2 constructs and examine changes in polyglutamylation patterns

• Co-localization analysis: Perform double immunofluorescence with TTLL2 antibodies and glutamylated tubulin antibodies to assess spatial relationships

• In vitro assays: Purify recombinant TTLL2 and assess its ability to add glutamate residues to tubulin substrates in a controlled biochemical setting

• Mass spectrometry: Analyze polyglutamylation patterns on tubulin before and after TTLL2 manipulation

These approaches, used in combination, can provide strong evidence for TTLL2's specific role in the polyglutamylation process and its effects on microtubule dynamics.

What methodologies are recommended for studying TTLL2 in neurodevelopmental disorders?

To investigate TTLL2's role in neurodevelopmental disorders, consider these methodological approaches:

• Patient-derived samples: Compare TTLL2 expression levels in samples from patients with neurodevelopmental disorders versus healthy controls using validated TTLL2 antibodies in Western blot, IHC, or IF

• Animal models: Develop and characterize TTLL2 knockout or mutant models to assess neurodevelopmental phenotypes

• Neuronal cultures: Manipulate TTLL2 expression in primary neuronal cultures and assess effects on neurite outgrowth, axon formation, and dendrite development

• High-resolution imaging: Use super-resolution microscopy with TTLL2 antibodies to examine subcellular localization in neuronal compartments

• Electrophysiology: Correlate TTLL2 expression or activity with neuronal function using patch-clamp recordings

• Genetic association studies: Analyze TTLL2 variants in patient cohorts and correlate with specific phenotypes

Each approach provides unique insights, and combining multiple methods strengthens the evidence for TTLL2's role in neurodevelopment and related disorders.

How can I conduct multiplex immunofluorescence studies with TTLL2 antibodies?

For multiplex immunofluorescence involving TTLL2 antibodies, consider this methodological framework:

• Antibody selection: Choose TTLL2 antibodies raised in a species different from other target antibodies to avoid cross-reactivity. TTLL2 antibodies are available with various conjugates including FITC, biotin, and others that can facilitate multiplexing

• Sequential staining: For challenging combinations, employ sequential staining with complete stripping or blocking between rounds

• Spectral unmixing: Use spectral imaging systems when fluorophores have overlapping emission spectra

• Optimization of antibody pairs:

• Controls: Include single-color controls for each antibody to confirm specificity and establish compensation settings

This approach enables visualization of TTLL2 alongside its substrates or interacting partners in the same sample, providing spatial context for functional studies.

How do I address non-specific binding issues with TTLL2 antibodies?

When encountering non-specific binding with TTLL2 antibodies, implement these troubleshooting strategies:

• Increase blocking stringency: Use 5% BSA instead of normal serum, or add 0.1-0.3% Triton X-100 to blocking buffer

• Optimize antibody concentration: Perform titration experiments to determine the minimum concentration providing specific signal

• Modify washing protocols: Increase washing duration and number of washes using PBS-T (0.1% Tween-20)

• Pre-absorb the antibody: Incubate with proteins from non-target tissues to remove cross-reactive antibodies

• Change blocking agents: Try different blocking agents (milk, BSA, normal serum, commercial blockers)

• Adjust fixation: Different fixatives (paraformaldehyde, methanol, acetone) may affect epitope accessibility

For Western blots specifically, higher dilutions of antibody (1:2000-1:10000) have been reported to be effective for TTLL2 detection while minimizing background .

What strategies can overcome detection sensitivity issues with TTLL2 antibodies?

To enhance detection sensitivity when working with TTLL2 antibodies:

• Signal amplification systems: Consider biotin-streptavidin amplification or tyramide signal amplification (TSA)

• Enhanced detection reagents: Use high-sensitivity substrates for HRP (e.g., SuperSignal West Femto)

• Sample enrichment: Immunoprecipitate TTLL2 before Western blot analysis

• Longer exposure times: For Western blots, extend exposure times with low-noise detection systems

• Optimize primary antibody incubation: Increase incubation time to overnight at 4°C

• Utilize conjugated antibodies: Direct conjugates (FITC, HRP) may provide enhanced sensitivity in some applications

• Consider polymer detection systems: HRP-polymer conjugated secondary antibodies can increase sensitivity in IHC applications

The choice of enhancement strategy should align with your specific application and the abundance of TTLL2 in your experimental system.

How can TTLL2 antibodies be used to investigate microtubule dynamics in cancer progression?

Investigating microtubule dynamics in cancer using TTLL2 antibodies can follow these methodological approaches:

• Comparative expression analysis: Quantify TTLL2 levels across cancer stages using validated antibodies in tissue microarrays

• Live-cell imaging: Use cell-permeable fluorescently tagged TTLL2 antibody fragments to track dynamic changes in cancer cells

• Correlation with therapeutic response: Assess TTLL2 expression before and after treatment with microtubule-targeting chemotherapeutics

• Cancer cell migration studies: Inhibit TTLL2 function and measure effects on cancer cell migration, which depends on microtubule dynamics

• Co-localization with mitotic spindle: Perform double immunofluorescence with TTLL2 and mitotic markers in cancer cells

These approaches can provide insights into how altered TTLL2 activity contributes to cancer progression through modification of tubulin and subsequent changes in microtubule dynamics.

What considerations are important when using TTLL2 antibodies across different species?

When applying TTLL2 antibodies across species, consider these methodological points:

• Epitope conservation: Verify sequence homology of the antibody's target epitope across species of interest

• Cross-reactivity validation: Test antibodies on positive control samples from each species before experimental use

• Optimization by species: Different dilutions may be optimal for different species (typically more concentrated for less conserved regions)

• Species-specific positive controls: TTLL2 gene orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee and chicken species

• Selective antibodies: Choose antibodies raised against conserved regions for cross-species applications

The table below summarizes sequence identity of TTLL2 across common research species:

This information helps researchers select appropriate antibodies for comparative studies across species.