GUCD1 Antibody

What is GUCD1 and what cellular functions does it perform?

GUCD1 (guanylyl cyclase domain containing 1) is a highly conserved protein that contains a guanylyl cyclase 2 domain, which characterizes a family of proteins capable of catalyzing the conversion of GTP to guanosine 3',5'-cyclic monophosphate (cGMP) and pyrophosphate. The human GUCD1 gene spans 3619 bp with a coding sequence of 723 bp and is comprised of 5 introns and 6 exons located on chromosome 22. GUCD1 has been identified as a protein upregulated during liver regeneration and may have roles in regulating normal and abnormal cell growth in the liver . Studies have shown remarkable conservation of GUCD1 across species, with 99% identity in mouse, rat, and human amino acid sequences, suggesting an evolutionarily important function .

What are the validated applications for GUCD1 antibodies in research?

According to product information, commercially available anti-GUCD1 antibodies have been validated for specific research applications including:

These applications enable researchers to detect and visualize GUCD1 protein expression in both cell lysates and intact cells . When selecting a GUCD1 antibody, researchers should verify that it has been specifically validated for their intended application to ensure reliable and reproducible results.

What is the subcellular localization of GUCD1 protein?

Immunolocalization studies have consistently demonstrated that GUCD1 exhibits a predominantly cytoplasmic localization. This has been confirmed through multiple experimental approaches:

• Immunofluorescence analysis of COS-1 cells transfected with both native and tagged GUCD1 constructs showed diffuse distribution with prevalent localization within the cytoplasm .

• Endogenous GUCD1 protein in HepG2 hepatocarcinoma cells similarly displayed predominantly cytoplasmic localization .

• Cell fractionation experiments have further validated these findings .

Understanding the cytoplasmic localization of GUCD1 is crucial for designing experiments and interpreting results, particularly when using subcellular fractionation techniques or co-localization studies.

What are the recommended protocols for GUCD1 antibody storage and handling?

For optimal antibody performance and longevity, researchers should follow these evidence-based storage recommendations:

• Short-term storage: Store at +4°C for immediate use or short-term preservation (days to weeks) .

• Long-term storage: Store at -20°C for extended preservation (months to years) .

• Avoid repeated freeze-thaw cycles as this can compromise antibody activity.

• If possible, aliquot the antibody solution before freezing to minimize freeze-thaw cycles.

• Follow manufacturer-specific recommendations, as formulation buffers may vary.

Improper storage can lead to antibody degradation, aggregation, or loss of specificity, potentially compromising experimental results and requiring additional validation steps.

How should researchers optimize Western blot protocols for GUCD1 detection?

When using anti-GUCD1 antibodies for Western blot analysis, consider these optimization strategies:

• Expected band size: GUCD1 protein appears as a specific band of approximately 27 kDa in Western blots of endogenous protein . Tagged versions may run at higher molecular weights (e.g., a fusion protein with myc tag ran at 36 kDa) .

• Sample preparation:

  • Total cell lysates are suitable for GUCD1 detection

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying potential post-translational modifications

• Controls:

  • Use lysates from cells known to express GUCD1 (e.g., HepG2, ARO cells) as positive controls

  • Include negative controls when possible

  • Consider using recombinant GUCD1 as a standard

• Blocking: 5% non-fat dry milk in TBST is generally suitable, but optimization may be required

• Troubleshooting high background:

  • Increase blocking time or concentration

  • Reduce primary antibody concentration

  • Increase washing stringency

These recommendations are based on published research that successfully detected GUCD1 in various cell lines and tissues .

What considerations are important for immunocytochemistry applications with GUCD1 antibodies?

For successful immunocytochemistry (ICC) experiments detecting GUCD1, researchers should consider:

• Fixation method:

  • Paraformaldehyde (4%) fixation has been successfully used in published studies of GUCD1

  • Over-fixation may mask epitopes; optimize fixation time

• Permeabilization:

  • Since GUCD1 is predominantly cytoplasmic, effective membrane permeabilization is essential

  • Triton X-100 (0.1-0.5%) is commonly used and effective for cytoplasmic proteins

• Expected staining pattern:

  • Diffuse cytoplasmic staining is the expected pattern for GUCD1

  • Nuclear exclusion should be observed

• Signal amplification:

  • Consider signal amplification methods for low-abundance detection

  • Tyramide signal amplification may improve sensitivity while maintaining specificity

• Controls:

  • Include cells with GUCD1 knockdown or knockout as negative controls

  • Consider co-staining with markers of different cellular compartments

When imaging, focus on cytoplasmic regions as GUCD1 has been shown to have predominantly cytoplasmic localization in both overexpression studies and endogenous protein detection .

How can researchers validate the specificity of GUCD1 antibodies?

Validating antibody specificity is critical for reliable results. For GUCD1 antibodies, consider these approaches:

• Genetic validation:

  • Use GUCD1 knockout or knockdown models (siRNA, CRISPR) to confirm signal loss

  • Overexpression studies using tagged GUCD1 constructs can confirm co-localization with antibody signal

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide to block specific binding

  • Signal should be reduced or eliminated if the antibody is specific

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of GUCD1

  • Concordant results increase confidence in specificity

• Cross-species reactivity:

  • GUCD1 is highly conserved (99% identity in mouse, rat, and human)

  • Consistent detection across species supports specificity

• Expected molecular weight verification:

  • GUCD1 should appear at approximately 27 kDa in Western blots

  • Tagged versions will show appropriate size shifts

A comprehensive validation approach using multiple methods provides the strongest evidence for antibody specificity and experimental reliability.

What factors affect GUCD1 protein detection in different experimental contexts?

Several factors can influence GUCD1 detection, leading to experimental variability:

• Post-translational modifications:

  • Research has shown discrepancies between GUCD1 mRNA and protein levels in certain cell lines, suggesting post-translational regulation

  • GUCD1 interacts with NEDD4-1 (E3 ubiquitin protein ligase), which controls its stability through ubiquitination

• Cell-type specific expression:

  • GUCD1 expression varies across cell types, with higher expression observed in certain cancer cell lines (SK-MEL23, HepG2, Jurkat, and ARO cells)

  • Expression may be regulated differently in normal vs. transformed cells

• Growth conditions:

  • Cell cycle stage may affect expression levels, particularly given GUCD1's association with proliferative processes

  • Serum levels and cell density can influence expression

• Sample preparation:

  • Protein extraction methods affect yield and integrity

  • Buffer composition can influence epitope accessibility

• Detection system sensitivity:

  • Enhanced chemiluminescence (ECL) vs. fluorescence-based detection

  • Signal amplification methods may be necessary for low abundance detection

Understanding these factors allows researchers to design appropriate controls and interpret discrepancies across experiments or between different detection methods.

How is GUCD1 expression regulated during liver regeneration and what research methods best capture these changes?

GUCD1 was initially identified through screening of a regenerating liver cDNA library, indicating its upregulation during liver regeneration . To study GUCD1 regulation in this context, researchers should consider:

• Temporal expression analysis:

  • GUCD1 is upregulated early in liver regeneration

  • Time-course experiments are essential to capture dynamics

  • Both mRNA (qRT-PCR) and protein (Western blot) should be measured

• Partial hepatectomy model:

  • The standard 70% partial hepatectomy model in rodents has been used successfully to study GUCD1

  • Collection of liver samples at multiple timepoints post-surgery (6h, 24h, 48h, 72h)

• Cell cycle correlation:

  • GUCD1 modulation occurs during cell cycle progression in vitro

  • Consider synchronizing cells and measuring GUCD1 at different cell cycle stages

• Interaction partners during regeneration:

  • GUCD1 interacts with NEDD4-1, affecting its stability

  • Co-immunoprecipitation during different regeneration phases may reveal dynamic interactions

• Functional studies:

  • siRNA knockdown or CRISPR/Cas9 knockout of GUCD1 in hepatocytes

  • Assess effects on proliferation, cell cycle progression, and regenerative capacity

These approaches can help elucidate GUCD1's role in the complex process of liver regeneration and potentially in pathological conditions like hepatocellular carcinoma.

What is known about GUCD1's interaction with NEDD4 and how can researchers investigate this relationship?

GUCD1 interacts directly with NEDD4-1 (E3 ubiquitin protein ligase neural precursor cell expressed, developmentally downregulated gene 4), which controls GUCD1 stability through ubiquitination . To investigate this relationship:

• Interaction verification methods:

  • Yeast two-hybrid assays have successfully identified the GUCD1-NEDD4 interaction

  • Co-immunoprecipitation can confirm interaction in mammalian cells

  • Proximity ligation assay (PLA) can visualize interactions in situ

• Ubiquitination assessment:

  • Ubiquitination assays with HA-tagged ubiquitin

  • Proteasome inhibitors (MG132) to assess degradation pathway

  • Site-directed mutagenesis of potential ubiquitination sites

• Functional significance:

  • NEDD4 knockdown/knockout to assess effects on GUCD1 levels

  • Correlation of NEDD4 and GUCD1 levels across tissues and disease states

  • Impact on GUCD1's role in proliferation

• Structural basis:

  • Domain mapping to identify interaction interfaces

  • Potential for therapeutic targeting

Understanding this interaction may provide insights into post-translational regulation of GUCD1 and potential intervention points for modulating GUCD1 activity in pathological conditions.

What evidence exists for GUCD1's role in hepatocellular carcinoma and other cancers?

Research has revealed several lines of evidence suggesting GUCD1's involvement in cancer:

• Expression in hepatocellular carcinoma (HCC):

  • High-level expression of GUCD1 transcripts observed in livers from patients with HCC

  • Potential biomarker or therapeutic target

• Expression in cancer cell lines:

  • Increased expression of GUCD1 mRNA in multiple cancer cell lines compared to normal livers

  • Highest expression detected in SK-MEL23 (melanoma), HepG2 (liver), Jurkat (T-cell leukemia), and ARO (thyroid) cells

• Correlation with proliferation:

  • GUCD1 modulation occurs during cell cycle progression in vitro

  • Association with liver regeneration suggests roles in controlled proliferation

• Research approaches to investigate cancer roles:

  • Tissue microarray analysis of GUCD1 in tumor vs. adjacent normal tissue

  • Correlation with clinical parameters (staging, prognosis)

  • Functional studies in cancer cell lines (proliferation, migration, invasion)

  • In vivo tumor models with GUCD1 modulation

These findings suggest GUCD1 may have roles in carcinogenesis or tumor progression, particularly in HCC, though more research is needed to establish causal relationships and mechanistic details.

Why do discrepancies occur between GUCD1 mRNA and protein expression levels?

Research has identified notable discrepancies between GUCD1 mRNA and protein levels in certain cell lines, suggesting complex post-transcriptional and post-translational regulation . Several mechanisms may explain these discrepancies:

• Post-translational modifications:

  • GUCD1 interacts with NEDD4-1, an E3 ubiquitin ligase that controls GUCD1 stability

  • Ubiquitination may target GUCD1 for proteasomal degradation

  • Different cell types may have varying levels of NEDD4-1 or other regulatory proteins

• mRNA stability and translation efficiency:

  • GUCD1 mRNA has a relatively long 3'UTR region (2580 bp), which may contain regulatory elements affecting translation efficiency or mRNA stability

  • microRNA regulation may vary between cell types

• Methodological considerations:

  • Antibody affinity and epitope accessibility

  • Protein extraction efficiency

  • Detection method sensitivity

When designing experiments, researchers should:

• Measure both mRNA and protein levels when possible

• Consider protein half-life (proteasome inhibitors, cycloheximide chase)

• Investigate post-translational modifications (ubiquitination, phosphorylation)

• Examine regulatory pathways specific to the cell type being studied

Understanding these regulatory mechanisms may provide insights into GUCD1's function in normal and pathological conditions.

How should researchers analyze GUCD1 expression data from different tissue types?

When analyzing GUCD1 expression across different tissues, consider these methodological approaches:

• Reference gene selection:

  • Use multiple reference genes for qRT-PCR normalization

  • Validate reference gene stability across the specific tissues being compared

  • Consider tissue-specific reference genes when appropriate

• Protein normalization strategies:

  • Total protein normalization (Ponceau S, REVERT total protein stain) may be more reliable than single housekeeping proteins

  • Verify loading control stability across tissue types

• Comparative analysis framework:

  • Establish a baseline (e.g., normal liver for HCC studies)

  • Use fold-change relative to baseline rather than absolute values

  • Consider log transformation for wide expression ranges

• Statistical considerations:

  • Account for biological variability within tissue types

  • Use appropriate statistical tests based on data distribution

  • Consider multiple testing correction for large-scale analyses

• Integrated analysis:

  • Correlate GUCD1 expression with other markers (proliferation, cell cycle)

  • Consider pathway analysis rather than isolated gene expression

These approaches help ensure robust and reproducible analysis of GUCD1 expression across diverse tissue types and experimental conditions.

How can researchers differentiate between specific and non-specific binding in GUCD1 antibody applications?

Distinguishing specific from non-specific binding is critical for accurate interpretation of GUCD1 antibody experiments. Consider these approaches:

• Expected molecular weight verification:

  • GUCD1 protein appears as a specific band of approximately 27 kDa

  • Tagged versions (e.g., myc-tagged) run at predictably higher weights (approximately 36 kDa)

  • Multiple or unexpected bands may indicate non-specific binding or protein degradation

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide should abolish specific binding

  • Persistent signals likely represent non-specific binding

• Genetic approaches:

  • GUCD1 knockdown/knockout should reduce/eliminate specific signal

  • Overexpression should increase specific signal

  • Unchanged bands after these manipulations suggest non-specificity

• Cross-validation with multiple antibodies:

  • Different antibodies targeting different GUCD1 epitopes should yield consistent results

  • Discrepancies may indicate non-specific binding of one antibody

• Isotype control antibodies:

  • Same isotype (e.g., rabbit IgG) at same concentration

  • Controls for non-specific binding due to Fc receptor interactions or other isotype-specific effects

By implementing these approaches systematically, researchers can confidently distinguish genuine GUCD1 signals from artifacts, ensuring reliable and reproducible results.