DOCK7 Antibody, HRP conjugated

What is DOCK7 and why is it important in research?

DOCK7 (dedicator of cytokinesis 7) is a guanine nucleotide exchange factor (GEF) that activates small GTPases, particularly Rac1 and Cdc42, which are key regulators of cell migration and cytoskeletal rearrangement. It plays crucial roles in neuronal development and function, with expression primarily in neuronal cells but also detected across various tissues. DOCK7 contains conserved Dock Homology Region 1 (DHR1) and DHR2 domains, with DHR2 containing GEF activity that enables GDP-GTP exchange on target GTPases . The protein has been implicated in multiple signaling pathways including AKT and mTOR/S6K signaling, making it relevant to researchers studying neurological disorders and cancer biology .

What is the recommended starting dilution range for HRP-conjugated DOCK7 antibodies in Western blotting?

For HRP-conjugated DOCK7 antibodies in Western blotting applications, researchers should begin with dilutions in the range of 1:1000 to 1:5000, then optimize based on signal strength and background levels. This recommendation is based on the typical dilution ranges for unconjugated DOCK7 antibodies (1:5000-1:50000 for Western blotting) but adjusted for the direct HRP conjugation . The optimal dilution will depend on protein expression levels in your specific samples, with tissues known to have high DOCK7 expression (brain tissue, neuronal cell lines) potentially requiring higher dilutions. Since DOCK7 is a high molecular weight protein (observed at 238-243 kDa), ensure adequate separation with appropriate percentage gels (typically 6-8% acrylamide) and extended transfer times to allow complete protein transfer .

How should I validate the specificity of DOCK7 antibody in my experimental system?

Validating DOCK7 antibody specificity requires a multi-faceted approach. First, conduct positive and negative control experiments using tissues or cell lines with known DOCK7 expression profiles. Based on the search results, HeLa, Jurkat, HEK-293T, and brain tissues (human, mouse, rat) are suitable positive controls . For negative controls, utilize DOCK7 knockdown approaches through siRNA or shRNA techniques, which should demonstrate significant reduction in signal if the antibody is specific. The search results mention DOCK7 shRNA transgenic mice showing decreased expression in sciatic nerves .

For further validation, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody captures DOCK7 protein. Additionally, check for a single band of appropriate molecular weight (238-250 kDa) in Western blot applications, with potential slight variations due to post-translational modifications or splice variants (up to 7 different isoforms have been reported) . If developing complex experimental models, consider orthogonal validation by comparing results from at least two different DOCK7 antibodies targeting distinct epitopes to confirm consistent findings .

What are the optimal blocking conditions for minimizing background when using HRP-conjugated DOCK7 antibodies?

For optimal blocking conditions with HRP-conjugated DOCK7 antibodies, a systematic approach is necessary. Begin with 5% non-fat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20) as your standard blocking solution, which effectively blocks non-specific binding sites without interfering with specific antibody-antigen interactions. If background persists, experiment with alternative blocking agents including 3-5% BSA in TBST or commercial blocking solutions specifically designed for HRP-conjugated antibodies. For challenging samples with high background, conduct a blocking agent titration (3%, 5%, and 10% concentrations) and time optimization (30 minutes, 1 hour, and 2 hours at room temperature).

When using HRP-conjugated antibodies, it's particularly important to include adequate washing steps (at least 3 x 5 minutes with TBST) after blocking and antibody incubation to remove unbound reagents. Additionally, consider incorporating a 0.05-0.1% sodium azide-free blocking step, as sodium azide can inhibit HRP activity and reduce signal strength . For tissues with high endogenous peroxidase activity (such as brain tissue where DOCK7 is highly expressed), pre-treat samples with hydrogen peroxide solutions (0.3-3% H₂O₂) before blocking to quench endogenous activity and improve signal-to-noise ratio .

What detection systems provide optimal sensitivity for HRP-conjugated DOCK7 antibodies in immunoblotting?

For optimal sensitivity with HRP-conjugated DOCK7 antibodies in immunoblotting, enhanced chemiluminescence (ECL) systems offer the best balance of sensitivity and practicality. Standard ECL provides adequate detection for samples with moderate to high DOCK7 expression, while advanced ECL formulations (often marketed as "ECL Plus," "ECL Advance," or "SuperSignal West Femto") can increase sensitivity by 10-100 fold for detecting low abundance DOCK7 protein or when using higher antibody dilutions to conserve reagent.

When working with challenging samples or requiring quantitative analysis, consider these methodological optimizations: (1) Substrate incubation time should be standardized (typically 1-5 minutes) with longer exposures for weak signals; (2) For quantitative work, capture multiple exposure times to ensure measurements are taken within the linear detection range; (3) For extremely low abundance samples, DAB (3,3'-diaminobenzidine) colorimetric detection can sometimes yield lower background than chemiluminescence, though with reduced sensitivity; and (4) Modern fluorescent Western blot systems using secondary antibodies may provide superior quantitative data compared to HRP-based detection for some applications, though this would require unconjugated primary antibodies . For DOCK7 specifically, optimizing protein extraction from membrane and cytoskeletal fractions may be necessary given its cellular localization and interactions with membrane-associated GTPases .

What factors might cause false negative results when detecting DOCK7 with HRP-conjugated antibodies?

Several methodological factors can lead to false negative results when detecting DOCK7 with HRP-conjugated antibodies. First, inadequate protein extraction is a common issue since DOCK7 is a high molecular weight protein (238-250 kDa) that may require specialized extraction buffers containing stronger detergents like 1% SDS or 0.5% sodium deoxycholate to efficiently solubilize membrane-associated proteins . Second, inefficient protein transfer during Western blotting is problematic for large proteins like DOCK7; use lower percentage gels (6-8%), extend transfer times (minimum 2 hours), or employ specialized transfer systems designed for high molecular weight proteins.

Additionally, degradation of the HRP conjugate can occur due to improper storage or repeated freeze-thaw cycles. HRP-conjugated antibodies should be stored at -20°C with glycerol (typically 50%) to prevent freeze-thaw damage, and aliquoting is recommended to minimize repeated thawing . Other methodological considerations include: (1) incompatible fixation methods that may mask the epitope; (2) denaturation conditions that destroy the epitope structure; (3) insufficient antibody concentration, particularly for tissues with low DOCK7 expression; and (4) presence of sodium azide or other HRP inhibitors in buffers, which directly interferes with the enzymatic activity of HRP conjugates .

How can I resolve multiple bands or unexpected molecular weights when detecting DOCK7?

Resolving multiple bands or unexpected molecular weights when detecting DOCK7 requires systematic troubleshooting. First, verify whether the additional bands represent genuine DOCK7 isoforms, as up to 7 different isoforms have been reported . Compare your pattern with published literature on DOCK7 detection in similar samples. To distinguish between specific and non-specific bands, perform validation experiments using DOCK7 knockdown controls - bands that disappear or significantly decrease in intensity after knockdown likely represent specific DOCK7 detection .

For methodological optimization: (1) Adjust sample preparation by testing different lysis buffers, adding additional protease inhibitors, and ensuring samples remain cold throughout processing to prevent degradation; (2) Modify blocking conditions by testing alternative blocking agents (5% milk vs. 3-5% BSA) which may reduce non-specific binding; (3) Titrate antibody concentration, as too high concentrations can increase non-specific binding; (4) Increase washing stringency by extending wash times or adding 0.1-0.5% SDS to wash buffers to reduce non-specific interactions; and (5) For HRP-conjugated antibodies specifically, ensure the detection substrate is fresh and the exposure time is optimal, as overexposure can reveal weak non-specific bands . If multiple bands persist after optimization, consider using immunoprecipitation followed by Western blotting to enrich for DOCK7 before detection .

How should I store and handle HRP-conjugated DOCK7 antibodies to maintain optimal activity?

Proper storage and handling of HRP-conjugated DOCK7 antibodies is crucial for maintaining their activity. Store the antibody at -20°C in buffer containing 50% glycerol and 0.02% sodium azide (though note that sodium azide inhibits HRP activity and must be removed before use) . Avoid repeated freeze-thaw cycles by preparing small working aliquots upon first thaw. When using the antibody, always maintain cold chain by keeping it on ice or at 4°C during experiments. Never vortex the antibody solution as this can denature both the antibody and the HRP conjugate; instead, mix by gentle inversion or flicking.

For long-term stability, protect HRP-conjugated antibodies from light exposure and oxidizing agents. The shelf-life is typically one year after shipment when properly stored . Before each use, centrifuge the antibody vial briefly to collect the solution at the bottom of the tube. When diluting, use freshly prepared buffers free of sodium azide or other peroxidase inhibitors. For working solutions, antibody stability can be extended by adding stabilizing proteins such as 0.1-1% BSA. If diminished activity is observed over time, this may indicate degradation, and fresh antibody should be used rather than simply increasing concentration, which could introduce non-specific binding .

How can I use DOCK7 antibodies to investigate its role in AKT/mTOR signaling pathways?

Investigating DOCK7's role in AKT/mTOR signaling requires multiple complementary approaches using DOCK7 antibodies. Start with co-immunoprecipitation (Co-IP) experiments to detect protein-protein interactions between DOCK7 and key pathway components. The search results demonstrate that DOCK7 associates with mTOR, TSC1, TSC2, and AKT in a multiprotein complex called DockTOR . For Co-IP experiments, use 0.5-4.0 μg of DOCK7 antibody per 1.0-3.0 mg of total protein lysate . Follow with Western blotting to detect both DOCK7 and its binding partners.

For functional studies, combine DOCK7 knockdown approaches with phospho-specific antibodies against AKT (particularly at Ser473, an mTORC2 site) and S6K to monitor pathway activation states. The search results indicate that Dock7 preserves AKT phosphorylation by protecting it from dephosphorylation and promotes mTOR/S6K activation during cellular stress . Establish rescue experiments by re-expressing wild-type DOCK7 or domain mutants (particularly DHR1 and DHR2 domains) after knockdown to identify critical regions for signaling activities.

Immunofluorescence with DOCK7 antibodies (dilution 1:50-1:500) can reveal subcellular co-localization with mTOR pathway components under different conditions (e.g., growth factor stimulation versus serum starvation) . For mechanistic insights, combine these approaches with pharmacological inhibitors of the pathway (e.g., Rapamycin, Torin, MK2206) to determine epistatic relationships between DOCK7 and downstream effectors .

What methodological approaches can reveal DOCK7's role in neuronal development using antibody-based techniques?

Investigating DOCK7's role in neuronal development requires multiple antibody-based methodological approaches. Immunohistochemistry and immunofluorescence (using 1:50-1:500 dilution) can map DOCK7 expression patterns throughout developmental stages in neuronal tissues . The search results indicate that DOCK7 is expressed mainly in neuronal cells and is localized to axons, growth cones, and neuron projections . Co-staining with markers for neuronal differentiation stages can reveal temporal expression patterns.

For functional studies, combine DOCK7 antibody staining with knockdown approaches. The search results mention DOCK7 shRNA transgenic mice showing enhanced myelin thickness, suggesting DOCK7 negatively regulates Schwann cell differentiation and myelination onset . Complementary in vitro studies using primary neuronal cultures with DOCK7 knockdown or overexpression can help elucidate its role in axon formation, neurite outgrowth, and polarization.

For mechanistic insights, investigate DOCK7's GEF activity toward Rac1 and Cdc42 using pull-down assays with GST-PAK-CRIB domain to detect active GTPases, followed by immunoblotting. Co-immunoprecipitation experiments (using 0.5-4.0 μg antibody per 1.0-3.0 mg lysate) can identify developmental stage-specific binding partners . Live imaging combined with DOCK7 antibody injection or expression of dominant-negative constructs can visualize real-time effects on neuronal morphogenesis and migration. These methodological approaches together can provide comprehensive insights into DOCK7's neuronal functions .

How can DOCK7 antibodies be applied to investigate its role in cancer biology?

DOCK7 antibodies can be applied to investigate its role in cancer biology through multiple methodological approaches. Begin with expression profiling using tissue microarrays and immunohistochemistry to compare DOCK7 levels across tumor types and stages versus normal tissues. The search results indicate that triple-negative breast cancer cells exhibit higher DOCK7 protein expression compared to other breast cancer subtypes . Follow with subcellular localization studies using confocal microscopy and fractionation experiments with Western blotting (recommended dilution 1:5000-1:50000) to detect potential mislocalization in cancer cells .

For functional studies, combine DOCK7 knockdown approaches with assays measuring cancer hallmarks. The search results show that DOCK7 knockdown decreased anchorage-independent growth in multiple cancer cell lines (triple-negative MDA-MB-231, receptor-positive SK-BR-3 and MCF7 breast cancer cells, HeLa cervical cancer cells, and A549 lung cancer cells) . DOCK7 appears particularly important for cancer cell survival during stress conditions like serum starvation, rather than proliferation or invasion .

Mechanistically, use DOCK7 antibodies for co-immunoprecipitation (0.5-4.0 μg antibody per 1.0-3.0 mg lysate) to identify cancer-specific interacting partners . The search results reveal that DOCK7 forms a multiprotein complex (DockTOR) including Cdc42, AKT, mTOR, TSC1/2, and Rheb, which sustains AKT phosphorylation and promotes mTOR/S6K activation during cellular stress . This provides a mechanistic basis for DOCK7's role in cancer cell survival. For translational relevance, correlate DOCK7 expression with clinical outcomes and treatment responses in patient samples using antibody-based detection methods .

How do results from DOCK7 HRP-conjugated antibodies compare with other detection methods?

Comparing results from DOCK7 HRP-conjugated antibodies with other detection methods reveals important methodological considerations. HRP-conjugated antibodies typically provide higher sensitivity in Western blotting compared to alkaline phosphatase (AP) conjugates, particularly for low abundance proteins, but may show higher background in some tissues with endogenous peroxidase activity like liver. When compared to fluorescent detection systems, HRP-conjugated antibodies offer advantages in cost and stability, but fluorescent methods typically provide better linear dynamic range for quantification and multiplexing capabilities.

For DOCK7 detection specifically, comparison studies should systematically evaluate: (1) detection threshold using serial dilutions of recombinant DOCK7 protein; (2) signal-to-noise ratio across different sample types; (3) reproducibility through technical replicates; and (4) correlation with functional assays or known DOCK7 expression patterns. When comparing with non-antibody methods like mRNA quantification, consider that DOCK7 protein levels may not always correlate with transcript levels due to post-transcriptional regulation.

Methodologically, researchers should design side-by-side comparisons using identical samples processed in parallel with different detection methods. For DOCK7, which exists in multiple isoforms, different detection methods may have varying abilities to distinguish between isoforms or detect post-translational modifications . Each result should be validated with appropriate controls, including DOCK7 knockdown samples, to confirm specificity across detection platforms .

What experimental design is necessary for studying DOCK7 interactions with small GTPases using antibody-based techniques?

Studying DOCK7 interactions with small GTPases requires a carefully designed experimental approach using antibody-based techniques. Begin with co-immunoprecipitation (Co-IP) experiments using DOCK7 antibodies (0.5-4.0 μg per 1.0-3.0 mg lysate) to pull down endogenous complexes, followed by immunoblotting for Rac1, Cdc42, and other potential GTPase partners . The search results indicate that DOCK7 functions as a GEF for small GTPases, particularly Rac1 and Cdc42 .

For determining the activation state of bound GTPases, combine IP with active GTPase pull-down assays using GST-PAK-CRIB domain constructs that specifically bind GTP-loaded (active) GTPases. To examine the spatial distribution of interactions, perform proximity ligation assays (PLA) using DOCK7 antibodies paired with GTPase-specific antibodies, which will generate fluorescent signals only when proteins are within 40 nm of each other.

For functional studies, design experiments that manipulate DOCK7 levels (knockdown/overexpression) followed by GTPase activity assays. The search results mention that DHR2 domain contains a GEF domain that can activate either Cdc42 or Rac1, and an allosteric binding site for activated Cdc42 . Create domain mutants of DOCK7 (particularly DHR1 and DHR2) to identify regions critical for GTPase interactions.

Live-cell imaging techniques can be combined with FRET-based GTPase activity biosensors to visualize DOCK7-dependent GTPase activation in real-time. Include appropriate controls: (1) GTPase-deficient mutants; (2) constitutively active GTPase variants; and (3) other GEF proteins to demonstrate specificity of DOCK7 interactions .

How can phospho-specific antibodies be combined with DOCK7 antibodies to investigate signal transduction mechanisms?

Combining phospho-specific antibodies with DOCK7 antibodies enables comprehensive investigation of signal transduction mechanisms through multiple methodological approaches. Begin with sequential immunoprecipitation experiments: first immunoprecipitate DOCK7 using specific antibodies (0.5-4.0 μg per 1.0-3.0 mg lysate), then probe the immunoprecipitates with phospho-specific antibodies against potential phosphorylation sites on DOCK7 or co-precipitated proteins . The search results indicate that DOCK7 interacts with phosphorylated AKT (particularly at Ser473) and is involved in the mTOR/S6K signaling pathway .

For temporal analysis of signaling events, design time-course experiments following stimulation (e.g., growth factors, stress conditions) and collect samples at defined intervals. Process these samples in parallel for both total DOCK7 detection and phosphorylation status of pathway components. The results show that stresses like serum deprivation promote the assembly of a DockTOR complex that sustains AKT phosphorylation and promotes mTOR/S6K activation .

Mechanistic studies should incorporate pharmacological inhibitors of kinases or phosphatases along with DOCK7 manipulation (overexpression/knockdown). The search results demonstrate that treatment with Torin (pan mTOR inhibitor) or MK2206 (AKT inhibitor) blocked growth in soft agar, while Rapamycin (mTORC1 inhibitor) partially blocked transformation, suggesting involvement of multiple signaling branches .

For spatial resolution, perform multi-color immunofluorescence microscopy using DOCK7 antibodies (1:50-1:500 dilution) together with phospho-specific antibodies to visualize co-localization of DOCK7 with activated signaling components in subcellular compartments . Include appropriate controls, such as phosphatase treatment of samples, to confirm specificity of phospho-antibody detection.