OBSCN Antibody, HRP conjugated

What is obscurin (OBSCN) and why is it significant in molecular research?

Obscurin is a giant protein (720-870 kDa) initially identified in striated muscles where it plays essential roles in normal muscle formation and function. Recent evidence has demonstrated that variants of obscurin ("obscurins") are also expressed in non-muscle tissues, contributing to distinct cellular processes. Importantly, dysfunction or abrogation of obscurins has been implicated in several pathological conditions, including cardiac hypertrophy and cancer . The OBSCN gene encodes this protein, which has been recognized as a potent tumor suppressor in pancreatic epithelial cells. Loss of obscurin function has been shown to accelerate pancreatic cancer progression and metastasis, thereby shortening survival . These multifaceted roles make obscurin an important target for various research applications, particularly in understanding cytoskeletal organization, cellular signaling, and disease mechanisms.

What are the key benefits of using HRP-conjugated antibodies in obscurin research?

Horseradish peroxidase (HRP)-conjugated antibodies offer several methodological advantages when studying obscurin:

• Enhanced sensitivity: HRP is a 44kDa glycoprotein with 4 lysine residues for conjugation that produces colored, fluorimetric, or luminescent derivatives of labeled molecules, allowing for detection and quantification with high sensitivity .

• Signal amplification: HRP has a high turnover rate that enables the generation of strong signals in a relatively short time span, making it particularly useful for detecting low-abundance proteins like obscurin in complex samples .

• Stability and cost-effectiveness: HRP is smaller, more stable, and less expensive than other popular enzyme alternatives, making it ideal for secondary antibody conjugation in various research applications .

• Versatility: HRP-conjugated antibodies can be used across multiple applications including Western blotting, ELISA, and immunohistochemistry, providing flexible experimental design options .

How should researchers evaluate the specificity of OBSCN antibodies before experimental use?

When evaluating OBSCN antibody specificity, researchers should implement a multi-step validation approach:

• Analyze epitope targeting: Confirm which domain of obscurin the antibody recognizes. For example, some antibodies specifically target the first immunoglobulin domain of obscurin-A/obscurin-B (amino acids 1-100) .

• Conduct cross-reactivity testing: Verify species reactivity and potential cross-reactivity with related proteins. For example, OBSCN antibodies from certain vendors are tested for reactivity with human, mouse, and rat samples .

• Perform knockout/knockdown validation: Use cells with OBSCN gene silencing through techniques like shRNA (e.g., using constructs targeting human OBSCN gene sequences such as 5'-AGAGGCAGGAGCCAGTGCCACACTGAGCT-3' and 5'-CTTGAGGATGCTGGAACTGTCAGTTTCCA-3') to confirm antibody specificity.

• Include positive and negative controls: Use tissues known to express high levels of obscurin (e.g., striated muscle) as positive controls and non-expressing tissues as negative controls.

• Compare multiple antibodies: When possible, validate findings using different antibodies targeting distinct epitopes of the obscurin protein.

What are the optimal conditions for using OBSCN antibody in Western blot applications?

Optimizing Western blot protocols for OBSCN detection requires careful consideration of the protein's large size (720-870 kDa) and potential isoform diversity:

• Sample preparation:

  • Use protease inhibitor cocktails during protein extraction to prevent degradation

  • Consider low percentage (3-5%) SDS-PAGE gels for better separation of high molecular weight proteins

  • Extend running time at lower voltage to improve resolution of large proteins

• Transfer conditions:

  • Implement wet transfer methods rather than semi-dry for large proteins

  • Use lower current (30-40mA) for extended periods (overnight) at 4°C

  • Consider adding 0.1% SDS to transfer buffer to facilitate movement of large proteins

• Antibody dilution and incubation:

  • For primary OBSCN antibodies, start with manufacturer's recommended dilution (typically 1:20-1:30 for immunoblotting)

  • For HRP-conjugated secondary antibodies, dilutions between 1:10,000-1:50,000 are often effective

  • Extend primary antibody incubation to overnight at 4°C for improved sensitivity

• Detection optimization:

  • Use enhanced chemiluminescent (ECL) substrates with extended signal duration

  • Adjust exposure times to optimize signal-to-noise ratio for this large protein

• Controls:

  • Include positive control samples from tissues known to express obscurin

  • Consider using OBSCN knockdown samples as negative controls

How can researchers effectively use OBSCN antibody, HRP conjugated in ELISA applications?

For optimal ELISA performance with OBSCN antibody, HRP conjugated:

• Coating parameters:

  • When using recombinant OBSCN protein as a standard, coat plates with 1-5 μg/ml in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C for optimal protein binding

• Blocking optimization:

  • Use 3-5% BSA or non-fat dry milk in TBST or PBS-T buffer

  • Block for 1-2 hours at room temperature to minimize non-specific binding

• Sample preparation:

  • Prepare serial dilutions of samples to ensure measurements fall within the linear range

  • Consider pre-clearing complex samples to reduce matrix effects

• Antibody application:

  • For directly HRP-conjugated OBSCN antibodies, start with 1 μg/mL concentration and optimize based on signal intensity

  • Incubate for 1-2 hours at room temperature or overnight at 4°C

• Detection and quantification:

  • Use TMB (3,3',5,5'-tetramethylbenzidine) substrate for HRP detection

  • Measure absorbance at 450nm after stopping the reaction with sulfuric acid

  • Calculate protein concentration using a standard curve with recombinant OBSCN protein

• Validation:

  • Include internal controls across plates to monitor inter-assay variation

  • Perform spike-and-recovery experiments to validate ELISA performance in different sample matrices

What troubleshooting approaches can resolve weak or absent signals when using OBSCN antibody, HRP conjugated?

When encountering weak or absent signals with OBSCN antibody:

• Protein extraction assessment:

  • Verify extraction efficiency for this large protein using alternative extraction buffers with increased detergent concentrations

  • Confirm protein integrity by staining with total protein stains before immunodetection

• Antibody functionality verification:

  • Test antibody with positive control samples known to express OBSCN

  • Verify antibody activity using dot blot of recombinant OBSCN protein

  • Check if HRP enzyme activity is inhibited by cyanides, azides, or sulfides in your buffers

• Detection system enhancement:

  • Increase antibody concentration incrementally (e.g., try 1:10 dilution if manufacturer recommends 1:20)

  • Extend incubation times for primary antibody (overnight at 4°C)

  • Use more sensitive detection reagents (e.g., femto-level ECL substrates)

  • Consider signal amplification systems such as biotin-streptavidin

• Reduce epitope masking:

  • Include additional antigen retrieval steps if using fixed tissues

  • Test different detergents or denaturing conditions that may expose hidden epitopes

• Buffer optimization:

  • Ensure proper buffer composition including appropriate concentrations of BSA (15 mg/mL) in antibody diluent similar to commercial preparations

  • Adjust salt concentration in wash buffers (typically 0.25M sodium chloride) to reduce non-specific binding

How can OBSCN antibody, HRP conjugated be used to study the role of obscurin in cancer progression?

To investigate obscurin's role in cancer progression using HRP-conjugated antibodies:

• Expression profiling methodology:

  • Perform immunohistochemistry on tissue microarrays containing normal tissue, primary tumors, and metastases

  • Quantify expression levels using digital image analysis of HRP-generated signal intensity

  • Compare OBSCN expression between matched tumor and normal samples (research has shown significantly reduced obscurin levels in human PDAC biopsies compared to matched controls)

• Functional analysis protocols:

  • Generate stable knockdown cell lines using OBSCN-specific shRNA constructs as described in previous studies (e.g., using sequences: 5'-AGAGGCAGGAGCCAGTGCCACACTGAGCT-3' and 5'-CTTGAGGATGCTGGAACTGTCAGTTTCCA-3')

  • Assess cell migration, invasion, and cytoskeletal organization changes using immunofluorescence with HRP-conjugated antibodies

  • Analyze cell signaling pathways affected by obscurin loss, particularly RhoA signaling which has been implicated in obscurin-regulated cancer progression

• In vivo model development:

  • Establish xenograft models using OBSCN-knockdown and control cells

  • Monitor tumor growth and metastasis formation

  • Perform ex vivo analysis of tumors using OBSCN antibody, HRP conjugated to assess protein expression in tumor microenvironment

• Correlation with clinical outcomes:

  • Analyze OBSCN expression patterns in patient samples using HRP-based immunohistochemistry

  • Correlate expression levels with survival data and other clinical parameters

  • Develop multivariate models incorporating OBSCN expression as a potential prognostic marker

What methodological approaches can be used to investigate obscurin's interaction with RhoA signaling?

To study obscurin-RhoA signaling interactions:

• Co-immunoprecipitation protocols:

  • Use anti-OBSCN antibodies to pull down protein complexes

  • Probe for RhoA and related signaling components in immunoprecipitates

  • Perform reciprocal co-IP with RhoA antibodies and probe for obscurin

• RhoA activity assays:

  • Implement GTP-RhoA pull-down assays in control and OBSCN-knockdown cells

  • Compare active RhoA levels using HRP-conjugated secondary antibodies in Western blots

  • Quantify differences in RhoA activation following obscurin manipulation

• Visualization of co-localization:

  • Perform dual immunofluorescence for obscurin and RhoA

  • Use HRP-conjugated secondary antibodies with tyramide signal amplification for improved detection

  • Analyze co-localization using confocal microscopy and quantitative image analysis

• Functional rescue experiments:

  • Express constitutively active or dominant negative RhoA constructs in OBSCN-knockdown cells

  • Assess whether RhoA modulation rescues phenotypes associated with obscurin loss

  • Document changes in cytoskeletal organization and cell migration

• Downstream signaling analysis:

  • Examine the activation status of RhoA effectors (ROCK, mDia) in the presence and absence of OBSCN

  • Use phospho-specific antibodies and HRP-conjugated secondaries to detect activation of downstream kinases

  • Create signaling profiles that characterize the obscurin-RhoA pathway

How can researchers differentiate between obscurin isoforms using antibody-based techniques?

To distinguish between obscurin isoforms:

• Epitope-specific antibody selection:

  • Choose antibodies targeting unique domains present in specific isoforms

  • For instance, use antibodies against the first immunoglobulin domain (aa1-100) to detect both obscurin-A and obscurin-B

  • Select antibodies against C-terminal domains to differentiate between major isoforms

• Western blot optimization for isoform separation:

  • Use low percentage gradient gels (3-8%) to effectively separate high molecular weight isoforms

  • Extend electrophoresis time to achieve better resolution between similar-sized variants

  • Implement extended transfer times (overnight at low current) for complete transfer of large proteins

• RT-PCR analysis to complement protein detection:

  • Design primers specific to unique regions of each isoform

  • Correlate mRNA expression with protein detection using HRP-conjugated antibodies

  • Quantify relative abundance of different isoforms across tissue types

• Immunofluorescence localization patterns:

  • Compare subcellular localization of different isoforms using domain-specific antibodies

  • Document differential localization patterns (e.g., M-band localization in striated muscle cells)

  • Correlate localization with function in different cellular compartments

What strategies can enhance the detection sensitivity of low-abundance OBSCN protein?

To improve detection of low-abundance OBSCN:

• Signal amplification systems:

  • Implement tyramide signal amplification (TSA) with HRP-conjugated antibodies

  • Use biotin-streptavidin systems to enhance HRP signal intensity

  • Consider polymer-based detection systems with multiple HRP molecules per antibody

• Sample enrichment techniques:

  • Perform immunoprecipitation to concentrate OBSCN before detection

  • Use subcellular fractionation to isolate compartments with higher OBSCN concentration

  • Consider native protein complex isolation to maintain structural integrity

• Substrate optimization:

  • Select highly sensitive chemiluminescent substrates with femtomolar detection limits

  • Optimize substrate incubation time and concentration

  • Use digital imaging systems with cooling capabilities for extended exposure times

• Background reduction strategies:

  • Implement more stringent blocking protocols using 3-5% BSA or non-fat dry milk

  • Include additional washing steps with optimized buffer compositions

  • Use specialized low-background detection reagents designed for HRP systems

• Instrumentation considerations:

  • Utilize cooled CCD camera systems for detecting weak signals

  • Implement iterative exposure protocols to capture optimal signal range

  • Consider advanced microscopy techniques like TIRF for improved signal-to-noise ratio

What are the best approaches for multiplexing OBSCN detection with other protein markers?

For effective multiplexing strategies:

• Sequential detection protocols:

  • Perform sequential immunodetection with stripping between antibodies

  • Use HRP inactivation steps (e.g., sodium azide treatment) between detections

  • Implement different substrates for each round of HRP detection (chromogenic, fluorescent, chemiluminescent)

• Antibody selection for multiplexing:

  • Choose antibodies from different host species to avoid cross-reactivity

  • Use directly conjugated primary antibodies to eliminate secondary antibody cross-reactivity

  • Validate antibody combinations prior to experimental use using known positive controls

• Spectral separation techniques:

  • When using fluorescent detection, select fluorophores with minimal spectral overlap

  • Implement linear unmixing algorithms to separate overlapping signals

  • Use sequential scanning approaches for confocal microscopy applications

• Statistical colocalization analysis:

  • Calculate Pearson's correlation coefficient for quantitative colocalization assessment

  • Implement Manders' overlap coefficient for proportion-based colocalization analysis

  • Use specialized software (ImageJ plugins, CellProfiler) for automated colocalization analysis

• Controls for multiplexing experiments:

  • Include single-stained controls for determining bleed-through

  • Implement isotype controls to assess non-specific binding

  • Use knockdown/knockout samples as negative controls for specificity validation

How should researchers optimize fixation and permeabilization conditions for OBSCN immunodetection in tissue samples?

For optimal OBSCN detection in tissues:

• Fixation protocol selection:

  • Compare different fixatives (4% paraformaldehyde, methanol, acetone) for optimal epitope preservation

  • Test fixation times to balance structural preservation with epitope accessibility

  • For OBSCN's large size, consider using lower fixative concentrations with longer incubation times

• Antigen retrieval optimization:

  • Implement heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Test enzymatic retrieval methods (proteinase K, trypsin) at various concentrations and incubation times

  • Optimize retrieval conditions specifically for the targeted OBSCN epitope

• Permeabilization strategy:

  • Test detergents of varying strengths (0.1-0.5% Triton X-100, 0.1% SDS, 0.1% Saponin)

  • Adjust permeabilization time based on tissue thickness and density

  • Consider freeze-thaw cycles for difficult-to-permeabilize samples

• Blocking optimization:

  • Use species-matched serum (5-10%) combined with BSA (1-3%)

  • Include additional blocking steps with unconjugated secondary antibodies

  • Test commercial blocking solutions specifically designed for HRP-based detection systems

• Signal development considerations:

  • For HRP-conjugated antibodies, optimize DAB development time to maximize signal while minimizing background

  • Implement controlled humidity during incubation steps to prevent edge effects

  • Consider using amplification systems specifically designed for tissue sections

Table 1: Recommended Dilutions and Applications for OBSCN Antibody, HRP Conjugated

RT = Room Temperature; ECL = Enhanced Chemiluminescence; TMB = 3,3',5,5'-Tetramethylbenzidine; DAB = 3,3'-Diaminobenzidine; TSA = Tyramide Signal Amplification; FFPE = Formalin-Fixed Paraffin-Embedded

Table 2: Comparison of Common Secondary Antibodies for OBSCN Detection

WB = Western Blot; ELISA = Enzyme-Linked Immunosorbent Assay; IHC = Immunohistochemistry; FLISA = Fluorescence-Linked Immunosorbent Assay