GUSBP1 Antibody

What is GUSBP1 and how does it differ from GUSB?

GUSBP1 (GUSB pseudogene 1) refers to "Putative inactive beta-glucuronidase-like protein SMA3," alternatively known as "Putative beta-glucuronidase-like protein" or "SMA3" . It is related to but distinct from GUSB (beta-glucuronidase), which is a lysosomal hydrolase involved in the degradation of glucuronic acid-containing glycosaminoglycans. While GUSB is a tetrameric glycoprotein with enzymatic activity that plays crucial roles in degrading dermatan and keratan sulfates , GUSBP1 is considered a pseudogene product and likely lacks enzymatic activity.

The key differences between these molecules include:

What applications are GUSBP1 antibodies validated for?

Commercial GUSBP1 antibodies are primarily validated for Western Blot (WB) applications, with some also validated for ELISA . The application scope includes:

• Western Blot (WB): The most common application, with recommended dilutions typically ranging from 1:500-1:2000

• ELISA: Some antibodies are validated for this application

• Cell Line Testing: Validated on human cell lines like K562

It's important to note that unlike antibodies against the related GUSB protein, which are often validated for multiple applications including immunohistochemistry and immunofluorescence , GUSBP1 antibodies currently have a more limited range of validated applications.

What are the optimal storage and handling conditions for GUSBP1 antibodies?

For maximum stability and performance, GUSBP1 antibodies require specific storage and handling conditions:

• Long-term storage: Store at -20°C for up to one year

• Short-term/frequent use: Store at 4°C for up to one month

• Avoid: Repeated freeze-thaw cycles

• Buffer composition: Typically provided in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Aliquoting: For frequent use, creating small aliquots is recommended to avoid repeated freeze-thaw cycles

When handling the antibody for experiments, it's advisable to keep it on ice when removed from storage and to return it to appropriate temperature conditions promptly after use.

How should GUSBP1 antibodies be validated before use in experimental protocols?

Proper validation of GUSBP1 antibodies is crucial for experimental reliability. Recommended validation approaches include:

• Positive control testing: Using cell lines known to express GUSBP1, such as K562 cells

• Specificity verification: Testing on multiple cell lines or tissues to confirm consistent banding patterns

• Blocking peptide assessment: Using the immunogenic peptide to compete with antibody binding, which should eliminate specific signals

• Knockdown/knockout verification: Testing on samples where GUSBP1 has been silenced or deleted via RNA interference or CRISPR-Cas9

• Dilution optimization: Testing a range of antibody dilutions to determine optimal signal-to-noise ratio

• Cross-reactivity testing: Evaluate potential cross-reactivity with the related GUSB protein

A customer question in the search results highlights the importance of validation: researchers inquired about using a GUSBP1 antibody validated for human tissues in pig tissues, indicating the need for cross-species validation .

What are the optimal experimental conditions for detecting GUSBP1 using Western blot?

Optimizing Western blot protocols for GUSBP1 detection requires attention to several key parameters:

Sample Preparation:

• Lyse cells in RIPA or other appropriate buffer containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Load 20-50 μg of total protein per lane

Gel Electrophoresis and Transfer:

• Use 10-15% polyacrylamide gels (considering GUSBP1's ~15 kDa size)

• Transfer to PVDF membrane at 100V for 60-90 minutes in cold transfer buffer

• Verify transfer efficiency with Ponceau S staining

Antibody Incubation:

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary anti-GUSBP1 antibody at 1:500-1:2000 dilution overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Wash 3-5 times with TBST

Signal Development:

• Use enhanced chemiluminescence (ECL) detection system

• Optimize exposure time to prevent signal saturation

• Consider using digital imaging systems for quantification

How does GUSBP1 relate to GUSB function in disease models?

While direct evidence of GUSBP1's role in disease is limited in the search results, its relationship to GUSB warrants investigation given GUSB's established disease associations:

• Cancer and Immunotherapy Resistance: GUSB overexpression has been linked to primary resistance to anti-PD1 therapy in hepatocellular carcinoma (HCC) by downregulating PD-L1 expression through miR-513a-5p promotion . Investigating whether GUSBP1 modulates these pathways could reveal potential regulatory relationships.

• Pseudo-enzymatic Role: As a pseudogene product, GUSBP1 might function as a competitive inhibitor or regulator of GUSB activity, potentially influencing glycosaminoglycan metabolism.

• Regulatory RNA Function: Some pseudogenes function as regulatory RNAs. GUSBP1 might regulate GUSB expression through RNA-RNA interactions or by sequestering microRNAs targeting GUSB.

Research approaches to investigate these relationships could include:

• Correlation analyses of GUSBP1 and GUSB expression in disease tissues

• Functional studies using GUSBP1 overexpression or knockdown

• Investigation of GUSBP1's effect on miR-513a-5p and PD-L1 expression pathways

What challenges exist in distinguishing GUSBP1 from GUSB in experimental settings?

Distinguishing GUSBP1 from GUSB presents several experimental challenges:

• Sequence Similarity: As a pseudogene product, GUSBP1 shares sequence similarity with GUSB, potentially leading to cross-reactivity of antibodies or primers.

• Molecular Weight Discrimination: The substantial difference in molecular weight (GUSBP1: ~15 kDa vs. GUSB: ~75-78 kDa) provides one distinguishing feature in Western blot applications .

• Expression Level Differences: GUSB is widely expressed in various tissues, while GUSBP1 expression patterns are less characterized, potentially making detection more challenging.

• Antibody Specificity: Ensuring antibodies specifically recognize GUSBP1 without cross-reacting with GUSB requires thorough validation.

Strategies to address these challenges include:

• Using antibodies raised against regions with the greatest sequence divergence

• Performing parallel knockdown experiments for both genes

• Including both positive controls (overexpression constructs) in experimental designs

• Using gene-specific primers designed to unique regions for RT-PCR applications

How can researchers troubleshoot non-specific binding when using GUSBP1 antibodies?

Non-specific binding is a common challenge when working with antibodies against less-characterized targets like GUSBP1. Effective troubleshooting approaches include:

• Optimization of Blocking Conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time or concentration

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Antibody Dilution Optimization:

  • Test serial dilutions to identify optimal concentration (1:500-1:2000 recommended range)

  • Prepare antibody in fresh blocking buffer

• Enhanced Washing Protocols:

  • Increase number of washes (5-6 washes of 5-10 minutes each)

  • Use higher Tween-20 concentration in wash buffers (0.1-0.2%)

  • Consider adding low salt (150 mM NaCl) to wash buffers

• Sample Preparation Modifications:

  • Ensure complete protein denaturation (boil samples for 5-10 minutes)

  • Use fresh reducing agents in sample buffer

  • Filter lysates to remove debris

• Validation Controls:

  • Include competing peptide controls

  • Use GUSBP1 knockdown/knockout samples as negative controls

  • Test antibody against recombinant GUSBP1 and GUSB to assess specificity

What is the potential relationship between GUSBP1 and resistance to immunotherapy treatments?

While the search results don't directly address GUSBP1's relationship with immunotherapy resistance, the established role of GUSB provides a framework for investigation:

Research has shown that GUSB overexpression in hepatocellular carcinoma leads to primary resistance to anti-PD1 therapy by downregulating PD-L1 expression through promotion of miR-513a-5p . Given GUSBP1's relationship to GUSB, several research hypotheses emerge:

• Regulatory Interaction: GUSBP1 might regulate GUSB expression or activity, indirectly influencing immunotherapy responses.

• miRNA Pathway Involvement: If GUSBP1 affects miR-513a-5p levels (similar to or opposing GUSB's effect), it could influence PD-L1 expression and immunotherapy outcomes.

• Biomarker Potential: GUSBP1 expression levels could serve as a biomarker for predicting immunotherapy response, complementing GUSB assessment.

Research methodologies to explore these hypotheses include:

• Analysis of GUSBP1 expression in responders versus non-responders to immunotherapy

• In vitro studies modulating GUSBP1 expression and assessing effects on PD-L1 expression

• Investigation of relationships between GUSBP1, GUSB, and miR-513a-5p expression

What methodologies are recommended for studying GUSBP1 function across different cellular contexts?

Investigating GUSBP1 function requires a multifaceted approach:

• Expression Modulation:

  • RNA interference (siRNA/shRNA) for transient or stable knockdown

  • CRISPR-Cas9 gene editing for complete knockout

  • Overexpression using expression vectors with appropriate tags (e.g., Myc, FLAG)

• Functional Assays:

  • Proliferation assays (CCK-8, EdU incorporation) to assess growth effects

  • Migration and invasion assays (transwell, wound healing) to evaluate motility

  • GUSB activity assays to investigate potential regulatory effects on the enzymatic function

• Molecular Interaction Studies:

  • Co-immunoprecipitation to identify protein binding partners

  • RNA immunoprecipitation to detect RNA-protein interactions

  • Proximity ligation assays to visualize protein interactions in situ

• Localization Studies:

  • Immunofluorescence to determine subcellular localization

  • Cell fractionation followed by Western blot to confirm compartmentalization

  • Live-cell imaging with fluorescently tagged GUSBP1

• Pathway Analysis:

  • RNA-seq following GUSBP1 modulation to identify affected pathways

  • Protein array or mass spectrometry to detect changes in protein expression/modification

  • MicroRNA profiling to investigate potential regulatory RNA functions

• Disease Model Integration:

  • Analysis in patient-derived xenografts or organoids

  • Correlation studies in patient samples across disease stages

  • Manipulation in model organisms where appropriate

How do different fixation and preparation methods affect GUSBP1 antibody performance?

The optimal fixation and preparation methods for GUSBP1 detection depend on the application:

For Western Blot applications:

• Sample preparation typically involves cell lysis using RIPA or other appropriate buffers with protease inhibitors

• Protein denaturation with SDS and heat (95-100°C for 5-10 minutes)

• Addition of reducing agents (β-mercaptoethanol or DTT) to disrupt disulfide bonds

For potential immunohistochemistry applications:

• One customer inquiry specifically asked about using GUSBP1 antibody on paraffin-embedded sections and recommended fixation methods

• The technical support response suggested that "PFA is best to use for fixation because it has better tissue penetration ability" and noted that "PFA needs to be prepared fresh before use"

• The response also cautioned that "Long term stored PFA turns into formalin, as the PFA molecules congregate and become formalin"

These considerations highlight the importance of proper sample preparation and fixation methods for obtaining reliable results with GUSBP1 antibodies.

What are the considerations for designing experiments to study potential GUSBP1-GUSB interactions?

Investigating potential functional relationships between GUSBP1 and GUSB requires carefully designed experiments:

• Co-expression Analysis:

  • Quantify GUSBP1 and GUSB expression levels across multiple cell lines and tissues

  • Determine whether expression patterns are correlated, inversely related, or independent

  • Analyze public datasets (TCGA, GTEx) for expression correlation in normal and disease states

• Functional Relationship Studies:

  • Modulate GUSBP1 expression (overexpression or knockdown) and measure effects on:

    • GUSB mRNA and protein levels

    • GUSB enzymatic activity using fluorescent or colorimetric substrates

    • Subcellular localization of GUSB

  • Perform reciprocal experiments modulating GUSB and measuring effects on GUSBP1

• Competition Assays:

  • If GUSBP1 potentially functions as a competitive inhibitor of GUSB, design in vitro competition assays

  • Express and purify recombinant GUSBP1 and test its effect on purified GUSB activity

  • Develop cell-based assays with measureable GUSB activity endpoints

• Interaction Studies:

  • Co-immunoprecipitation using antibodies against either protein to detect physical interactions

  • Proximity ligation assays to visualize potential interactions in situ

  • FRET or BiFC approaches using fluorescently tagged versions of both proteins

How can researchers effectively compare and contrast different commercially available GUSBP1 antibodies?

Selecting the optimal GUSBP1 antibody requires systematic evaluation:

• Technical Specifications Comparison:

• Validation Data Assessment:

  • Review manufacturer-provided validation images

  • Examine published literature using specific antibody catalog numbers

  • Consider customer reviews and Q&A sections on manufacturer websites

• Experimental Validation:

  • Test multiple antibodies side-by-side using identical samples and protocols

  • Evaluate specificity using knockdown/knockout controls

  • Assess lot-to-lot consistency if purchasing the same antibody multiple times

• Technical Support:

  • Evaluate manufacturer responsiveness to technical inquiries

  • Review detailed protocols provided by manufacturers

  • Consider availability of blocking peptides for specificity confirmation

What emerging techniques might enhance GUSBP1 detection and functional analysis?

Several cutting-edge approaches show promise for advancing GUSBP1 research:

• Enhanced Antibody Technologies:

  • Single-domain antibodies (nanobodies) for improved specificity

  • Recombinant antibodies with reduced batch-to-batch variation

  • Bifunctional antibodies for simultaneous detection of GUSBP1 and GUSB

• Advanced Imaging Methods:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell imaging with fluorescently tagged GUSBP1

  • Mass cytometry for single-cell protein expression analysis in heterogeneous samples

• Functional Genomics Approaches:

  • CRISPR activation/inhibition for precise gene expression modulation

  • CRISPR base editing for introducing specific mutations

  • High-throughput screening to identify GUSBP1 regulators or effectors

• Structural Biology:

  • Cryo-EM or X-ray crystallography to determine GUSBP1 structure

  • In silico structural comparison with GUSB to identify functional differences

  • Structure-guided design of specific probes or inhibitors

How might GUSBP1 research contribute to understanding disease mechanisms?

GUSBP1 research has potential to advance understanding in several disease contexts:

• Cancer Biology:

  • Given GUSB's role in immunotherapy resistance , GUSBP1 might function as a regulator of cancer immune evasion

  • GUSBP1 expression patterns across cancer types could reveal diagnostic or prognostic biomarkers

  • Understanding GUSBP1-GUSB interactions might lead to novel therapeutic approaches

• Lysosomal Storage Disorders:

  • If GUSBP1 regulates GUSB activity, it could influence diseases related to glycosaminoglycan metabolism

  • GUSBP1 might compensate for or exacerbate GUSB deficiencies in certain genetic conditions

  • Targeting GUSBP1-GUSB interactions could potentially modulate disease severity

• Inflammation and Immune Response:

  • GUSB plays roles in extracellular matrix remodeling during inflammation

  • GUSBP1 might modulate these processes, affecting inflammatory response dynamics

  • The PD-L1/miR-513a-5p pathway affected by GUSB could also be influenced by GUSBP1

• Neurodegenerative Diseases:

  • The search results indicated neuroscience as a research area for GUSBP1

  • Lysosomal function is increasingly recognized as important in neurodegeneration

  • GUSBP1 might play unexplored roles in neuronal health and disease