GORASP1 Antibody

What is GORASP1 and why is it important in cellular research?

GORASP1 encodes GRASP65, a Golgi-associated peripheral protein that plays a crucial role in maintaining Golgi structure and function. GRASP65 is primarily involved in stacking Golgi cisternae, as demonstrated through numerous in vitro experiments . The protein is N-terminally myristoylated, which, along with its binding to the coiled-coil Golgi protein GM130, enables its attachment to cis- and medial-Golgi cisternae . GRASP65 has significant implications for Golgi reassembly during cell division, glycosylation processes, and mitotic progression. Recent research has identified the first human pathogenic variant in GORASP1, associating it with a neurodevelopmental disorder that includes neurosensory, neuromuscular, and skeletal abnormalities . This discovery underscores the importance of GORASP1 in human physiology and disease pathology, making it a critical target for antibody-based research.

What types of GORASP1 antibodies are available for research applications?

Researchers have access to a diverse array of GORASP1 antibodies with varying specifications suitable for different experimental applications:

• Host Species: GORASP1 antibodies are primarily produced in rabbit and mouse hosts .

• Clonality: Both monoclonal (e.g., clone 3G1, 5C5) and polyclonal antibodies are available .

• Reactivity: While most antibodies react with human GORASP1, some also cross-react with rat, mouse, dog, cow, guinea pig, horse, rabbit, monkey, and pig samples .

• Target Regions: Antibodies targeting different regions of GORASP1 are available, including those specific to amino acids 1-440 (full-length), 35-84, 91-119, 221-440, and 300-440 regions .

• Applications: These antibodies are validated for various techniques including Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Flow Cytometry (FACS), and ELISA .

When selecting a GORASP1 antibody, researchers should carefully consider which specifications best align with their experimental design, target species, and intended applications.

How should GORASP1 antibodies be validated prior to experimental use?

Proper validation of GORASP1 antibodies is essential for generating reliable experimental results. The validation process should include:

• Specificity Testing: Verify antibody specificity by comparing staining patterns in control samples versus samples lacking GORASP1. The recent study on GORASP1 variants demonstrated complete absence of protein detection in patient fibroblasts using both full-length and C-terminal specific antibodies, confirming antibody specificity .

• Multiple Detection Methods: Use complementary techniques such as Western blotting and immunocytochemistry. In the pathogenic variant study, researchers confirmed the absence of GRASP65 through both methods .

• Subcellular Localization Verification: Confirm proper Golgi localization by co-staining with established Golgi markers such as GM130. In control fibroblasts, GRASP65 showed perfect colocalization with GM130, indicating its cis-Golgi distribution .

• Positive and Negative Controls: Include appropriate controls in each experiment. For GORASP1, this might include GORASP1 knockout or knockdown models as negative controls .

• Cross-Reactivity Assessment: If working with non-human samples, verify species cross-reactivity as specified in the antibody documentation .

Proper validation ensures experimental reliability and reproducibility when working with GORASP1 antibodies.

What are the optimal conditions for Western blotting detection of GORASP1?

Successful Western blotting for GORASP1/GRASP65 requires careful optimization of several parameters:

• Sample Preparation: Total protein extraction should be performed using buffers containing protease inhibitors to prevent degradation of GRASP65, which has a molecular weight of approximately 65-70 kD .

• Antibody Selection: Consider using antibodies that recognize different epitopes of GRASP65. The study of the pathogenic GORASP1 variant utilized two different antibodies: one directed against the entire protein (GRASP65 FL) and another specifically recognizing the C-terminal part (GRASP65 C-ter) .

• Blocking Conditions: A 5-10% solution of non-fat dry milk or bovine serum albumin (BSA) in TBS-T is typically effective for blocking non-specific binding sites.

• Antibody Dilution: GORASP1 antibodies should be diluted according to manufacturer recommendations, typically in the range of 1:100 to 1:1000, depending on the specific antibody .

• Detection Method: Both chemiluminescence and fluorescence-based detection systems are suitable for GORASP1 Western blots, with the choice depending on the required sensitivity and quantification needs.

• Expected Results: In control samples, a single band with the expected size of 65-70 kD should be detected, whereas this band would be absent in samples lacking functional GRASP65 .

These optimized conditions help ensure specific and sensitive detection of GORASP1 in Western blotting applications.

How should immunocytochemistry protocols be optimized for GORASP1 detection?

Immunocytochemical detection of GORASP1 requires careful attention to fixation, permeabilization, and antibody incubation conditions:

These optimized protocols facilitate clear visualization of GRASP65 in its native cellular context.

How can GORASP1 antibodies be used to study Golgi structure and dynamics?

GORASP1 antibodies serve as powerful tools for investigating Golgi architecture and functional dynamics:

These applications demonstrate the utility of GORASP1 antibodies in advancing our understanding of Golgi biology.

How do GORASP1 knockout models help in understanding GRASP65 function?

GORASP1 knockout models provide valuable insights into the functions of GRASP65 in vivo:

• Generation of Knockout Models: Several approaches have been used to create GORASP1-deficient models, including conventional gene knockout in mice , CRISPR/Cas9 gene editing in cell lines , and siRNA-based depletion .

• Phenotypic Analysis: A GORASP1 mouse knockout model was crossed with a conditional knockout (cFlox) mouse expressing endogenous GORASP2 to study the effects of depleting both GRASP proteins . These models allow researchers to examine the consequences of GRASP65 loss on Golgi structure, glycosylation, and cellular functions.

• Organoid Studies: Small intestine budding organoids derived from GORASP1-deficient mice and treated with tamoxifen to deplete GORASP2 provided a system to study the combined effects of GORASP protein depletion .

• Cell Line Models: RPE cells with GORASP1 mutations introduced by CRISPR/Cas9 have been used to recapitulate phenotypes observed in patient fibroblasts, including glycosylation anomalies and mitotic delays .

• Validation of Antibody Specificity: GORASP1 knockout models serve as excellent negative controls for validating antibody specificity. The absence of GRASP65 signal in immunoblotting and immunocytochemistry experiments confirms antibody specificity .

Understanding the phenotypic consequences of GORASP1 knockout helps elucidate the protein's functions and potential roles in disease pathology.

How can GORASP1 antibodies help investigate glycosylation defects?

GORASP1 antibodies are valuable tools for studying the relationship between GRASP65 and glycosylation processes:

• Detection of Glycosylation Anomalies: Studies have shown that loss of GRASP65 leads to glycosylation defects, particularly hyposialylation . GORASP1 antibodies can be used in combination with glycosylation markers to correlate GRASP65 levels with glycosylation status.

• Analysis of N-Glycosylation Pathways: GORASP1 knockout mice exhibited defects in protein transport through the secretory pathway, particularly on the cis side, as well as N-glycosylation defects . Antibodies can help visualize the localization of glycosylation enzymes in relation to GRASP65.

• Assessment of Plasma Membrane Protein Glycosylation: Glycosylation of plasma membrane proteins was found impaired in HeLa cells depleted of GRASP65 by CRISPR/Cas9 . GORASP1 antibodies can be used in combination with cell surface biotinylation to analyze how GRASP65 deficiency affects membrane protein processing.

• Correlation with Disease Phenotypes: In the patient with the GORASP1 pathogenic variant, a decrease in terminal sialylation was observed . GORASP1 antibodies can help establish direct links between GRASP65 deficiency and specific glycosylation defects.

• Rescue Experiments: Reintroduction of wild-type GRASP65 in deficient cells followed by immunostaining can demonstrate whether glycosylation defects are directly caused by GRASP65 loss and can be rescued by its restoration.

These approaches enable detailed investigation of the mechanisms connecting GRASP65 function to proper glycosylation processes.

What is the role of GORASP1 in mitotic progression and how can antibodies help study this?

GORASP1 antibodies provide crucial insights into GRASP65's role in cell division:

• Mitotic Index Quantification: The absence of GRASP65 in patient fibroblasts was associated with an increased mitotic index . GORASP1 antibodies can be used alongside mitotic markers like Ki67 and phospho-histone H3 (PH3) to assess mitotic progression.

• Cell Cycle Phase Analysis: Studies revealed an excess of prometaphases and metaphases with polar chromosomes in GRASP65-deficient cells, suggesting a delay in the cell cycle . Immunofluorescence with GORASP1 antibodies combined with cell cycle markers can help characterize these defects.

• Spindle Dynamics Investigation: siRNA-based depletion of GRASP65 caused defects in spindle dynamics . GORASP1 antibodies used in combination with tubulin and pericentrin staining can reveal how GRASP65 influences spindle formation and function.

• Time-lapse Imaging: GORASP1 antibodies conjugated to fluorescent proteins can be used in live-cell imaging to track GRASP65 dynamics during mitosis in real-time.

• Phosphorylation Studies: GRASP65 undergoes phosphorylation during mitosis, which can be detected using phospho-specific antibodies in combination with general GORASP1 antibodies to understand how post-translational modifications regulate its function during cell division.

These methodologies help elucidate the complex role of GRASP65 in ensuring proper mitotic progression and genomic stability.

How can CRISPR/Cas9 gene editing be combined with GORASP1 antibody studies?

CRISPR/Cas9 technology offers powerful approaches for studying GORASP1 function when combined with antibody-based detection:

• Generation of Knockout Models: sgRNAs targeting exon 9 of GORASP1 can be designed using tools like Santa Cruz Tefor software and introduced into cells via electroporation . GORASP1 antibodies can then confirm successful protein depletion.

• Creation of Patient-Specific Mutations: The pathogenic variant identified in a patient (c.1170_1171del; p.Asp390Glufs*18) was recapitulated in RPE cells using CRISPR/Cas9, allowing researchers to study its effects in a controlled genetic background .

• Validation of Edited Clones: After generating individual colonies from electroporated cells, DNA sequencing confirms GORASP1 mutations, while immunostaining with GORASP1 antibodies verifies protein depletion .

• Phenotypic Characterization: GORASP1 antibodies enable detailed characterization of Golgi structure, glycosylation, and mitotic progression in gene-edited cells, helping establish direct causal relationships between specific mutations and cellular phenotypes .

• Structure-Function Analysis: CRISPR/Cas9 can be used to generate targeted mutations in different domains of GORASP1, with antibodies detecting the resulting proteins to correlate structural changes with functional outcomes.

This integration of gene editing with antibody-based detection provides a comprehensive approach to understanding GORASP1 biology.

How can GORASP1 antibodies contribute to understanding the first human GORASP1-related disorder?

The recent identification of the first human pathogenic variant in GORASP1 opens new avenues for clinical research where antibodies play a crucial role:

• Diagnostic Development: GORASP1 antibodies can help develop diagnostic tests for the newly described Golgipathy caused by GORASP1 mutations. In the reported case, antibody testing revealed a complete absence of GRASP65 protein despite normal mRNA levels .

• Genotype-Phenotype Correlation: The patient with homozygous GORASP1 variant (c.1170_1171del; p.Asp390Glufs*18) presented with a neurodevelopmental disorder combining neurosensory, neuromuscular, and skeletal abnormalities . GORASP1 antibodies can help correlate protein expression levels with symptom severity in additional patients.

• Pathophysiological Mechanisms: GORASP1 antibodies revealed that the patient's variant affects the C-terminal region of the serine/proline-rich (SPR) domain of GRASP65, resulting in a complete absence of the protein . This information helps understand the molecular basis of the disorder.

• Biomarker Development: By correlating GRASP65 levels with specific glycosylation defects and cell cycle abnormalities, GORASP1 antibodies can help identify biomarkers for disease progression and treatment response.

• Screening of Potential Therapeutics: GORASP1 antibodies can be used to monitor restoration of protein expression or function in cellular models treated with potential therapeutic compounds.

These applications demonstrate how GORASP1 antibodies contribute to translating basic research findings into clinical understanding and potential treatments.

What are the methodological considerations for studying GORASP1 variants in patient samples?

Investigating GORASP1 variants in patient samples requires careful methodological approaches:

• Sample Collection and Processing: Primary fibroblasts from skin biopsies provide a valuable resource for studying GORASP1 variants, as demonstrated in the first reported human case . These cells should be processed according to standard protocols for culture and analysis.

• Transcriptional Analysis: Quantitative PCR with primers distributed throughout the GORASP1 transcript can determine whether variants affect mRNA expression. In the reported case, no significant difference was observed between patient and control fibroblast mRNAs, indicating that the variant did not affect transcription .

• Protein Expression Analysis: Western blotting with antibodies recognizing different regions of GRASP65 (full-length and C-terminal specific) is essential for determining protein expression. In the reported case, no GRASP65 was detected with either antibody, indicating complete protein absence .

• Immunocytochemical Analysis: Immunostaining with GORASP1 antibodies, combined with Golgi markers like GM130, can reveal changes in protein localization or Golgi structure in patient cells .

• Functional Assays: Analysis of glycosylation status and mitotic progression in patient cells provides insights into the functional consequences of GORASP1 variants .

• Validation in Model Systems: Recapitulating patient variants in cellular models using CRISPR/Cas9 helps confirm that observed phenotypes are directly caused by the GORASP1 mutation .

These methodological considerations ensure rigorous analysis of GORASP1 variants in clinical research settings.