DLC1 Antibody

What applications are DLC1 antibodies validated for in research?

DLC1 antibodies have been validated for multiple research applications based on empirical data. The Proteintech DLC1 antibody (15460-1-AP) is validated for Western blot (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), immunoprecipitation (IP), and ELISA applications . Similarly, the Anti-DLC1 Antibody (A38474) from antibodies.com has been validated specifically for Western blot and immunohistochemistry applications .

For optimal experimental results, the following dilution ranges are recommended:

These recommendations should serve as starting points, as optimal antibody concentration may vary depending on sample type and experimental conditions. Researchers should perform titration experiments to determine the optimal concentration for their specific experimental setup.

What species reactivity do commercially available DLC1 antibodies demonstrate?

DLC1 antibodies exhibit cross-species reactivity that is valuable for comparative studies. The Proteintech DLC1 antibody (15460-1-AP) has been tested and confirmed to react with human, mouse, and rat samples . The Anti-DLC1 Antibody (A38474) shows reactivity with human and mouse samples . This cross-species reactivity indicates conservation of the epitope recognition regions across these mammalian species, making these antibodies suitable for studies using both human samples and common laboratory animal models.

How should DLC1 antibodies be stored and handled for optimal performance?

Proper storage and handling are critical for maintaining antibody activity and specificity. According to manufacturer guidelines, DLC1 antibodies should be stored at -20°C. The Proteintech DLC1 antibody (15460-1-AP) is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, and remains stable for one year after shipment . The Anti-DLC1 Antibody (A38474) is supplied at 1.0 mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺) at pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

To preserve antibody performance, researchers should:

• Avoid repeated freeze-thaw cycles

• Maintain sterile handling conditions

• Follow manufacturer-specific recommendations for aliquoting (though for Proteintech's antibody, aliquoting is unnecessary for -20°C storage)

• Note special formulations (some preparations may contain 0.1% BSA as a stabilizer)

What are the expected molecular weights for DLC1 protein detection by Western blot?

The detection of DLC1 protein by Western blot reveals multiple isoforms with distinct molecular weights. Although the calculated molecular weight for the main DLC1 protein is reported as 171 kDa , experimental evidence shows that antibodies frequently detect proteins at different sizes corresponding to specific transcripts:

• A 123 kDa protein derived from the 6.1 Kb transcript

• A 127 kDa protein corresponding to the 6.2 Kb transcript

• A theoretical 169.8 kDa protein from the 7.6 Kb transcript (rarely detected in practice)

Researchers have observed that the 127 kDa band sometimes appears as a doublet, suggesting possible post-translational modifications of this isoform . When performing Western blot analysis with DLC1 antibodies, researchers should anticipate detecting multiple bands representing these different isoforms, with band intensity varying based on tissue type and experimental conditions.

In which tissues is DLC1 protein expression most prominent?

DLC1 protein exhibits distinct tissue-specific expression patterns that vary by isoform. Western blot analyses using anti-DLC1 antibodies have revealed:

These tissue-specific expression patterns correlate with quantitative PCR and Northern blot analyses, providing cross-validation across different experimental techniques . This differential expression suggests tissue-specific roles for the various DLC1 isoforms and underscores the importance of selecting appropriate positive control tissues when validating antibody performance.

What are the recommended protocols for DLC1 antibody use in immunofluorescence studies?

For immunofluorescence studies, the following optimized protocol has been successfully implemented with DLC1 antibodies:

• Seed cells expressing GFP or GFP-tagged human DLC1 on glass coverslips and incubate for 24 hours

• Fix cells with 4% paraformaldehyde

• Permeabilize with 0.25% Triton X-100 in PBS

• Block with 5% goat serum in PBS

• Incubate with DLC1 primary antibody (1:100 dilution in PBS) at 4°C overnight

• Wash thoroughly with PBS

• Incubate with appropriate Alexa-conjugated secondary antibodies (1:250 dilution) for 1 hour

• For visualization of actin or nuclei, incubate with phalloidin (1:50) for 1 hour

• Wash thoroughly with PBS

• Mount with gel mounting solution

This protocol has been successfully employed to visualize DLC1 protein localization in cellular contexts and can be adapted based on specific experimental requirements. Positive staining has been reported in HepG2 cells , making this cell line a suitable positive control for protocol optimization.

How can researchers validate DLC1 antibody specificity in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable experimental results. For DLC1 antibodies, multiple complementary approaches are recommended:

• Genetic validation: Studies have demonstrated that homozygous Dlc1 gene-trapped (Dlc1^gt/gt) serum-free mouse embryo cells show significantly reduced levels of the 123 kDa DLC1 protein compared to heterozygous or wild-type cells (P < 0.001) . This approach provides strong validation of antibody specificity through genetic manipulation of the target protein.

• Isoform analysis: Using antibodies raised against different epitopes can help distinguish between DLC1 isoforms. For example, an antibody raised against amino acids 111-370 from exon 5 can detect proteins translated from the 6.1, 6.2, and 7.6 Kb transcripts, as this exon is common to all three isoforms .

• Tissue-specific controls: Include tissues known to express high levels of specific DLC1 isoforms as positive controls:

  • For the 123 kDa isoform: liver, placenta, and spleen

  • For the 127 kDa isoform: thymus, lung, testis, spleen, and liver

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to demonstrate that specific binding is blocked when the antibody binding sites are occupied.

What are the key considerations for antigen retrieval in DLC1 immunohistochemistry?

Effective antigen retrieval is essential for successful immunohistochemistry with DLC1 antibodies. Based on empirical testing:

• The primary recommended method is using TE buffer at pH 9.0

• An alternative approach uses citrate buffer at pH 6.0

Positive IHC staining has been reported in human spleen tissue and human liver tissue using these antigen retrieval methods . The choice between these methods may depend on tissue type, fixation protocol, and specific experimental requirements. Researchers should systematically compare both methods to determine optimal conditions for their particular tissue samples.

Additionally, when optimizing IHC protocols with DLC1 antibodies, factors to consider include:

• Fixation time and conditions

• Section thickness

• Blocking reagents to minimize background

• Incubation times and temperatures

• Detection system sensitivity

How can DLC1 antibodies be used to investigate RhoGAP activity in experimental systems?

DLC1 functions as a RhoGTPase activating protein (RhoGAP), and DLC1 antibodies serve as valuable tools for investigating its regulatory role in Rho signaling. Research has demonstrated that cells with reduced DLC1 protein levels, as confirmed by Western blot with DLC1 antibodies, exhibit increased RhoA activity along with altered cytoskeleton structure and enhanced cell motility .

Researchers can implement the following approaches using DLC1 antibodies:

• Correlation analysis: Assess the relationship between DLC1 protein levels (detected by Western blot) and RhoA activity (measured by pull-down assays) across different cell types or experimental conditions.

• Co-localization studies: Use immunofluorescence with DLC1 antibodies alongside RhoA staining to investigate their spatial relationship in cells under different conditions.

• Intervention assessment: Evaluate how treatments that modulate DLC1 expression or activity affect downstream RhoA signaling pathways and cellular phenotypes.

• Structure-function analysis: Combine DLC1 antibodies recognizing different domains with functional assays to determine how specific regions contribute to RhoGAP activity.

These approaches can provide mechanistic insights into how alterations in DLC1 expression or function impact Rho signaling and associated cellular processes.

What are the implications of detecting different DLC1 isoforms in cancer research?

The ability to detect and distinguish between DLC1 isoforms using specific antibodies has significant implications for cancer research. DLC1 was initially identified as a tumor suppressor gene frequently inactivated in hepatocellular carcinoma, and subsequent studies have revealed its importance in various malignancies.

Research utilizing DLC1 antibodies has demonstrated:

• Isoform-specific expression patterns: Different DLC1 isoforms show tissue-specific expression, with the 123 kDa protein highly expressed in liver, placenta, and spleen, while the 127 kDa protein shows high expression in thymus, lung, testis, spleen, and liver .

• Altered expression in experimental models: In gene-trapped mouse embryonic cells (Dlc1^gt/gt), specific reduction of the 123 kDa band was observed, along with an increase in a cross-reacting 127 kDa band, suggesting possible compensatory mechanisms .

• Functional consequences: Cells with reduced DLC1 protein levels exhibit increased RhoA activity and altered cytoskeletal organization, phenotypes associated with cancer cell behavior .

This knowledge can guide cancer researchers in:

• Profiling DLC1 isoform expression across tumor types and stages

• Investigating correlations between specific isoform loss and disease progression

• Assessing restoration of DLC1 expression as a potential therapeutic approach

• Studying interactions between DLC1 and other molecules in cancer signaling networks

What are common issues encountered when using DLC1 antibodies and how can they be addressed?

Researchers working with DLC1 antibodies may encounter several challenges that can be systematically addressed:

• Multiple bands in Western blot:

  • Issue: Detection of multiple bands (123 kDa, 127 kDa, and possibly others)

  • Solutions:

    • Use positive controls with known DLC1 isoform expression

    • Optimize gel percentage for better separation

    • Consider longer running times for better resolution of closely spaced bands

    • Use isoform-specific antibodies when available

• Difficulty detecting larger isoforms:

  • Issue: Some researchers report inability to detect the predicted 169.8 kDa protein from the 7.6 Kb transcript

  • Solutions:

    • Optimize protein extraction for large proteins

    • Use gradient gels (4-12%) for better separation

    • Modify transfer conditions (lower voltage, longer time)

    • Consider semi-dry transfer systems for large proteins

• Variable immunostaining results:

  • Issue: Inconsistent staining patterns across experiments

  • Solutions:

    • Compare different fixation methods

    • Test both recommended antigen retrieval approaches (TE buffer pH 9.0 and citrate buffer pH 6.0)

    • Optimize blocking conditions to reduce background

    • Validate antibody specificity in your specific tissue/cell type

How should researchers optimize DLC1 antibody concentration for specific applications?

Determining optimal antibody concentration is critical for achieving specific signal with minimal background. Based on empirical evidence, the following approach is recommended:

• For Immunoprecipitation (IP):

  • Start with 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

  • Perform titration experiments with increasing antibody amounts

  • Include appropriate controls (IgG control, input sample)

  • Validate results by Western blot of immunoprecipitated material

• For Immunohistochemistry (IHC):

  • Begin with the recommended dilution range (1:20-1:200)

  • Perform serial dilutions on known positive tissues (human spleen or liver for DLC1)

  • Assess signal-to-noise ratio at each dilution

  • Consider signal amplification systems for low-abundance targets

• For Immunofluorescence (IF)/ICC:

  • Start with the recommended dilution range (1:10-1:100)

  • Test on cell lines with known DLC1 expression (e.g., HepG2 cells)

  • Include proper negative controls (secondary antibody only, isotype control)

  • Optimize exposure settings to capture specific signal while minimizing background

For all applications, remember that optimal concentration may be sample-dependent . Systematic optimization through titration experiments is essential for achieving reproducible, high-quality results.