GORASP2 Antibody

What is GORASP2 and what are its primary functions?

GORASP2 is a key structural protein of the Golgi apparatus with a molecular weight of approximately 55 kDa. It serves multiple critical cellular functions:

• Forms and maintains the Golgi ribbon structure through interactions with GORASP1/GRASP65

• Facilitates assembly and membrane stacking of Golgi cisternae

• Mediates Golgi stack reformation after breakdown during mitosis and meiosis

• Regulates intracellular transport of specific transmembrane proteins

• Required for normal acrosome formation during spermiogenesis

• Mediates ER stress-induced unconventional trafficking of core-glycosylated proteins

• Functions in autophagy by facilitating autophagosome-lysosome fusion

What applications are GORASP2 antibodies validated for?

GORASP2 antibodies have been validated for multiple research applications:

When selecting an antibody, verify the validation data for your specific application to ensure optimal results .

How do I optimize Western blot detection of GORASP2?

For optimal GORASP2 detection by Western blot:

• Sample preparation:

  • Use lysis buffers containing protease and phosphatase inhibitors

  • Cell lines with confirmed expression: COLO 320, HeLa, Raji, A549, and HepG2 cells

  • Tissue samples: Brain, testis, and colon show good expression

• Technical considerations:

  • Expected molecular weight: 47 kDa (calculated), but typically appears at 55-59 kDa due to post-translational modifications

  • Some antibodies detect bands up to 72 kDa depending on the cell type

  • Recommend using gradient gels (4-12%) for better resolution

• Validation controls:

  • Include siRNA knockdown samples to confirm specificity

  • Use phospho-specific antibodies when studying phosphorylation events

How can I study GORASP2's role in autophagy using antibodies?

GORASP2 plays an unconventional role in autophagy that is regulated by its O-GlcNAcylation status:

• Experimental approach:

  • Culture cells in varying glucose concentrations to modulate O-GlcNAcylation

  • Use starvation conditions to induce autophagy

  • Perform co-immunoprecipitation between GORASP2 and LC3

• Key findings to investigate:

  • GORASP2 localizes to the autophagosome-lysosome interface under glucose deprivation

  • De-O-GlcNAcylated GORASP2 interacts with LC3 through a specific LC3-interacting region (LIR)

  • This interaction facilitates autophagosome-lysosome fusion

• Methodological considerations:

  • Use anti-GORASP2 antibodies in combination with autophagy markers (LC3, LAMP2)

  • Monitor changes in GORASP2 localization during autophagy induction

  • Employ proximity ligation assays to verify direct protein-protein interactions

How do phospho-specific GORASP2 antibodies help study Golgi dynamics?

Phosphorylation of GORASP2 is a key regulatory mechanism during cell division:

• Research applications:

  • Tracking Golgi fragmentation during mitosis

  • Studying GORASP2 regulation by the MKK/ERK pathway

  • Investigating Golgi reassembly after cell division

• Available tools:

  • Phospho-GRASP55/GORASP2 (Thr264) antibodies specifically recognize phosphorylated forms

  • These can distinguish active (phosphorylated) from inactive forms

• Experimental design:

  • Synchronize cells at specific cell cycle stages

  • Treat with kinase inhibitors or phosphatase inhibitors

  • Perform Western blot and immunofluorescence with phospho-specific antibodies

  • Compare with total GORASP2 antibodies to determine phosphorylation ratio

What are the considerations when studying GORASP2 in immunofluorescence?

For successful immunofluorescence with GORASP2 antibodies:

• Optimal protocol:

  • Fixation: 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilization: 0.1% Triton X-100 (5 minutes) - gentler detergents preserve Golgi structure

  • Blocking: 5% BSA (1 hour)

  • Primary antibody: Dilutions range from 1:50-1:500 (antibody-dependent)

  • Counterstain: Include markers for Golgi (GM130), microtubules, and nuclear DNA

• Expected pattern:

  • Perinuclear ribbon-like structure corresponding to the Golgi apparatus

  • During mitosis, expect dispersed punctate staining

  • Under autophagy conditions, partial co-localization with LC3-positive structures

• Controls:

  • siRNA knockdown to verify specificity (shown in validation data for ab211532)

  • Cell lines with confirmed expression patterns: U251, MCF7, HeLa, A431

How can I design co-immunoprecipitation experiments to identify GORASP2 binding partners?

For successful co-immunoprecipitation (co-IP) of GORASP2:

• Lysis conditions:

  • Use mild lysis buffers (0.5-1% NP-40 or Triton X-100)

  • Include protease and phosphatase inhibitors

  • Consider crosslinking for transient interactions

• Antibody selection:

  • Several antibodies are validated for IP, including 10598-1-AP

  • Pre-test antibody performance in your experimental system

• Known interactions to investigate:

  • GORASP2-LC3 interaction (enhanced during autophagy)

  • GORASP2-LAMP2 interaction (at the autophagosome-lysosome interface)

  • GORASP2-GORASP1 interaction (in Golgi stacking)

• Experimental strategy:

  • Perform IP under different cellular conditions (normal, starvation, ER stress)

  • Use reciprocal IP to confirm interactions

  • Follow with mass spectrometry to identify novel binding partners

How can I validate GORASP2 antibody specificity?

To ensure antibody specificity:

• Genetic approach:

  • Compare antibody signal in control vs. GORASP2-depleted samples (siRNA/shRNA)

  • Use CRISPR-Cas9 knockout cells as negative controls

• Biochemical approach:

  • Test multiple antibodies targeting different epitopes

  • Include blocking peptide competition assays

  • Verify if the observed molecular weight matches expectations

• Cellular approach:

  • Check for expected subcellular localization (perinuclear Golgi pattern)

  • Confirm reduced signal following GORASP2 depletion (see validation data from ab211532)

Why might I observe different molecular weights for GORASP2?

GORASP2 can appear at different molecular weights due to:

• Post-translational modifications:

  • Phosphorylation increases apparent molecular weight

  • O-GlcNAcylation affects migration pattern

  • Calculated weight: 47 kDa; Observed weight: 55-72 kDa

• Technical factors:

  • Gel percentage affects migration

  • Buffer systems can influence apparent size

  • Some antibodies may detect splice variants or processed forms

• Experimental validation:

  • Compare results with multiple antibodies targeting different regions

  • Include dephosphorylation treatments to confirm phosphorylation-dependent shifts

  • Use mass spectrometry to characterize the detected protein form

What species reactivity should I consider when selecting a GORASP2 antibody?

Most commercial GORASP2 antibodies show:

• Validated reactivity:

  • Human: Most antibodies show strong reactivity

  • Mouse and Rat: Many antibodies cross-react with rodent GORASP2

  • Pig: Some antibodies have demonstrated reactivity

• Predicted reactivity (based on sequence homology):

  • Zebrafish, Bovine, Horse, Sheep, Rabbit, Dog, Chicken, Xenopus

• Factors affecting cross-reactivity:

  • Immunogen sequence conservation across species

  • Antibody type (monoclonal vs. polyclonal)

  • Epitope accessibility in different applications

How can I study GORASP2 O-GlcNAcylation and its functional effects?

GORASP2 O-GlcNAcylation serves as a glucose-sensing mechanism:

• Detection methods:

  • Immunoprecipitate GORASP2 followed by Western blot with anti-O-GlcNAc antibodies

  • Use wheat germ agglutinin (WGA) lectin affinity purification

  • Employ mass spectrometry to identify specific modification sites

• Functional analysis:

  • Compare wild-type vs. O-GlcNAcylation-deficient GORASP2 mutants

  • Modulate cellular O-GlcNAcylation using OGT inhibitors or OGA inhibitors

  • Culture cells in varying glucose concentrations to alter O-GlcNAcylation levels

• Key findings:

  • De-O-GlcNAcylation allows GORASP2 to function in autophagy

  • O-GlcNAcylation status affects GORASP2-LC3 interaction

  • Non-O-GlcNAcylated GORASP2 promotes autophagosome-lysosome fusion

How can GORASP2 antibodies be used to study unconventional protein trafficking?

GORASP2 mediates ER stress-induced unconventional trafficking:

• Experimental setup:

  • Induce ER stress with tunicamycin or thapsigargin

  • Track cargo protein (e.g., CFTR) trafficking using surface biotinylation

  • Perform immunofluorescence co-localization with GORASP2 antibodies

• Methodological approach:

  • Use GORASP2 antibodies alongside markers for ER, Golgi, and plasma membrane

  • Perform time-course experiments after ER stress induction

  • Combine with GORASP2 knockdown/overexpression to establish causality

• Key findings to investigate:

  • GORASP2 mediates ER-to-plasma membrane trafficking that bypasses the Golgi

  • This pathway is activated under specific stress conditions

  • Post-translational modifications regulate GORASP2's role in this process

What new functions of GORASP2 are emerging in current research?

Recent studies have uncovered multiple non-canonical functions of GORASP2:

• Autophagy regulation:

  • Functions as a glucose sensor through O-GlcNAcylation

  • Facilitates autophagosome-lysosome fusion under nutrient stress

  • Interacts with both LC3 and LAMP2 to bridge these organelles

• Reproductive biology:

  • Required for normal acrosome formation during spermiogenesis

  • Promotes colocalization of JAM2 and JAM3 at contact sites between germ cells and Sertoli cells

  • Critical for male fertility

• Unconventional protein trafficking:

  • Mediates ER stress-induced Golgi-independent trafficking of core-glycosylated CFTR

  • May function as a molecular chaperone in specific trafficking pathways

These emerging functions present exciting new avenues for research using GORASP2 antibodies.

How can super-resolution microscopy enhance GORASP2 studies?

Super-resolution techniques offer significant advantages for GORASP2 research:

• Applications:

  • Resolving Golgi subdomains and GORASP2 distribution

  • Tracking GORASP2 dynamics during Golgi fragmentation and reassembly

  • Visualizing interactions with binding partners at nanometer resolution

• Technical considerations:

  • Choose highly specific antibodies with minimal background

  • For STORM/PALM: Ensure proper photoswitchable dye conjugation

  • For SIM: Use high-quality mounting media to prevent artifacts

  • For STED: Select dyes with appropriate depletion wavelength compatibility

• Validated antibodies:

  • Monoclonal antibodies like ab211532 have been successfully used in high-resolution microscopy

  • Multiple fluorescence images confirm perinuclear Golgi localization