GTPBP1 Antibody

What is GTPBP1 and why is it important in research?

GTPBP1 (GTP binding protein 1) is a translational GTPase involved in maintaining ribosomal integrity and ensuring proper translation, which is essential for cellular function. It plays a critical role in resolving paused ribosomes, particularly during tRNA deficiency, and functions as an important quality control mechanism during translation elongation . Recent research has linked bi-allelic variants in GTPBP1 to neurodegenerative and neurodevelopmental disorders, making it an important target for neurological research .

Which species reactivity is confirmed for commercially available GTPBP1 antibodies?

Most commercially available GTPBP1 antibodies demonstrate confirmed reactivity with human samples. Some antibodies, such as Proteintech's 16374-1-AP, have verified reactivity with human, mouse, and rat samples . When selecting an antibody, researchers should consult validation data for each specific antibody, as reactivity can vary between products and may be predicted based on sequence homology but not experimentally confirmed for all species .

What are the common applications for GTPBP1 antibodies?

GTPBP1 antibodies have been validated for several experimental applications:

Most antibodies require optimization for specific experimental conditions, and researchers are advised to titrate antibodies to obtain optimal results .

How should researchers design experiments to study GTPBP1's role in ribosome pausing?

To study GTPBP1's role in ribosome pausing, researchers should consider the following methodological approach:

• Ribosome profiling: This technique allows detection of ribosome occupancy at specific codons. For example, studies have shown increased ribosome occupancy at AGA codons in Gtpbp1 knockout mice .

• RNA sequencing: Combine with ribosome profiling to correlate pausing events with transcriptional changes. Previous studies identified 260 genes with both increased AGA ribosome occupancy and differential expression in Gtpbp1-deficient mice .

• Cell models: Compare wild-type cells with GTPBP1-knockout or knockdown cells, particularly under conditions of tRNA deficiency.

• Neuronal models: Given GTPBP1's role in neurodegeneration, primary neuronal cultures or brain tissue from model organisms can provide physiologically relevant contexts .

What control proteins and samples should be included when using GTPBP1 antibodies?

When designing experiments with GTPBP1 antibodies, the following controls should be considered:

• Loading controls: Standard loading controls such as vinculin (used at 1:20,000 dilution) have been employed in previous GTPBP1 studies .

• Negative controls:

  • GTPBP1 knockout or knockdown samples

  • Secondary antibody-only controls for immunohistochemistry

• Positive controls: Several studies have successfully used mouse skeletal muscle tissue and Jurkat cells as positive controls for GTPBP1 detection by Western blot .

• Related proteins: GTPBP2 can serve as a comparison point, as it's a paralog with similar functions but non-redundant roles .

How can researchers differentiate between GTPBP1 and GTPBP2 functions experimentally?

Despite being homologous proteins involved in ribosomal homeostasis, GTPBP1 and GTPBP2 have distinct non-redundant functions. To differentiate them experimentally:

• Comparative knockout studies: Both Gtpbp1-/- and Gtpbp2-/- mice show similar but not identical phenotypes. Analysis revealed 27 genes with significant differential expression between these two knockout models .

• mRNA degradation assays: GTPBP1 can stimulate exosomal degradation of mRNAs, unlike GTPBP2, providing a functional distinction between these proteins .

• Codon-specific pausing: While both proteins resolve AGA pauses, comparative ribosome profiling of knockout models can identify codon preferences specific to each protein .

• Expression pattern analysis: In situ hybridization with specific probes for Gtpbp1 and Gtpbp2 can differentiate their expression patterns in tissues, particularly in neuronal subtypes .

What are the key considerations when interpreting GTPBP1 antibody data in neurodegeneration studies?

When using GTPBP1 antibodies in neurodegeneration research, consider these critical factors:

• Neuronal subtype specificity: GTPBP1 effects vary among neuronal populations. For example, target genes like Sesn2, Slc7a1, and Chac1 are upregulated in both CA1 and DG neurons in mutant mice, whereas Ddr2 is only upregulated in CA1 neurons .

• Integrated stress response (ISR) activation: GTPBP1 deficiency activates the ISR pathway. Researchers should monitor eIF2α phosphorylation (p-eIF2α S51) levels, which increase in the cerebellum and hippocampus of GTPBP1-deficient mice .

• mTORC1 signaling interaction: GTPBP1 knockout affects mTOR signaling. Researchers should include assessment of mTORC1 pathway components, as decreased mTORC1 signaling contributes to increased neuronal death in GTPBP1-deficient models .

• Tissue fixation methods: For optimal immunohistochemistry results with GTPBP1 antibodies, researchers have successfully used 4% paraformaldehyde, 10% neutral buffered formalin, or Bouin's fixative depending on the downstream application .

How can researchers investigate GTPBP1's role in circadian rhythm regulation?

GTPBP1 has been identified as playing a role in the regulation of circadian mRNA stability . To investigate this function:

• Time-course experiments: Sample collection at different circadian times (e.g., every 4 hours across a 24-hour cycle).

• mRNA stability assays: Measure half-lives of circadian-regulated transcripts in the presence and absence of GTPBP1.

• RNA immunoprecipitation: Identify circadian-regulated mRNAs that directly interact with GTPBP1.

• Core clock gene expression: Monitor expression of core clock genes (e.g., Per1/2, Cry1/2, Bmal1, Clock) in GTPBP1-deficient models.

• Circadian behavior assays: For in vivo studies, assess whether GTPBP1 deficiency alters circadian locomotor activity patterns in model organisms.

What experimental approaches can detect GTPBP1-mediated ribosome quality control in disease models?

To investigate GTPBP1's quality control functions in disease contexts:

• Ribosome profiling in disease models: Compare ribosome pausing patterns between control and disease models with and without GTPBP1 manipulation.

• Polysome profiling: Assess changes in polysome/monosome ratios to evaluate global translation efficiency.

• Nascent peptide analysis: Use techniques like puromycin labeling to measure the production of nascent peptides.

• Genetic rescue experiments: In disease models with reduced GTPBP1 function, test if phenotypes can be rescued by GTPBP1 overexpression or by manipulating downstream pathways:

  • ISR inhibition/activation

  • mTORC1 modulation (e.g., using rapamycin, which has been used at 5 mg/kg in mouse studies)

• Patient-derived models: For human neurodevelopmental disorders linked to GTPBP1 variants, patient-derived cells can be used to assess translational defects .

What are the technical considerations for using GTPBP1 antibodies in co-immunoprecipitation studies?

For successful co-immunoprecipitation (co-IP) studies with GTPBP1:

• Antibody amount optimization: Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate for optimal results .

• Buffer considerations:

  • For RNA-binding proteins like GTPBP1, consider using RNase inhibitors in lysis buffers

  • Include GTP or non-hydrolyzable GTP analogs to preserve GTP-dependent interactions

• Control IPs:

  • IgG control from the same species as the GTPBP1 antibody

  • Parallel IP in GTPBP1-depleted cells

• Validation of interactions:

  • Confirm by reverse co-IP using antibodies against the interacting partners

  • Consider mass spectrometry to identify novel interacting proteins

• Sample preparation: Mouse skeletal muscle tissue has been successfully used for GTPBP1 immunoprecipitation studies and can serve as a positive control .

What are common issues with GTPBP1 antibody specificity and how can they be addressed?

When working with GTPBP1 antibodies, researchers may encounter specificity challenges:

• Cross-reactivity with GTPBP2: Due to sequence similarity between GTPBP1 and its paralog GTPBP2, antibodies may cross-react. Solutions include:

  • Using antibodies raised against divergent regions

  • Validating with knockout/knockdown controls

  • Performing side-by-side testing with known GTPBP1 and GTPBP2 antibodies

• Non-specific bands in Western blot: GTPBP1 has an expected molecular weight of 71-72 kDa . Additional bands may represent:

  • Post-translationally modified forms

  • Degradation products

  • Non-specific binding

• Validation approaches:

  • siRNA/shRNA knockdown

  • CRISPR/Cas9 knockout

  • Peptide competition assays using the immunizing peptide

How should researchers interpret conflicting data between GTPBP1 antibody results and genetic studies?

When antibody-based detection and genetic approaches yield conflicting results:

• Antibody epitope consideration: Check if genetic variants or manipulations affect the antibody epitope region. For example, some GTPBP1 antibodies target the C-terminal region (aa 578-608) , while others target regions within aa 550-650 .

• Protein vs. mRNA discrepancies: Consider post-transcriptional regulation of GTPBP1. Compare protein levels (antibody) with mRNA expression (qPCR, RNA-seq).

• Compensatory mechanisms: In genetic models, compensatory upregulation of related proteins (e.g., GTPBP2) may occur. Use antibodies against multiple related proteins to assess compensation.

• Technical validation:

  • Use multiple antibodies targeting different epitopes

  • Combine approaches (e.g., immunostaining with in situ hybridization)

  • Include positive and negative control samples in all experiments

• Species differences: Phenotypes may differ between species. For example, mouse GTPBP1 knockout showed no observable phenotype in early studies , while human GTPBP1 variants are linked to neurodevelopmental disorders .