eif3ja Antibody

Basic Research Questions

• What is eIF3J and what applications are available for eIF3J antibodies?

  eIF3J is one of the smallest subunits of eIF3, critical in 40S initiation complex assembly. It associates with the aminoacyl site and mRNA entry channel of the 40S ribosomal subunit . eIF3J antibodies can be applied in multiple experimental contexts:

  Application	Recommended Dilution
  Western Blotting	1:500-1:1000
  Immunoprecipitation	0.5-4.0 μg for 1.0-3.0 mg of total protein lysate
  Immunohistochemistry	1:20-1:200
  Immunofluorescence	1:200-1:800

  Importantly, eIF3J antibodies have been validated in multiple cell lines including HeLa, A549, HEK-293, Jurkat, K-562, MCF-7, and SKOV-3 cells .

• What species reactivity should be considered when selecting an eIF3J antibody?

  When designing cross-species experiments, researchers should carefully select antibodies with appropriate reactivity profiles:

  Antibody Source	Species Reactivity
  eIF3J Antibody #3261	Human, Mouse, Rat
  EIF3J Antibody (10439-1-AP)	Human (primary validation), Pig (cited)

  Cross-reactivity validation is essential before conducting comparative studies across species. Researchers should perform preliminary western blot analysis with positive controls from target species to confirm reactivity.

• How should eIF3J antibodies be stored to maintain optimal activity?

  Proper storage is critical for maintaining antibody performance:

  • Store at -20°C in aliquots to avoid freeze-thaw cycles

  • Most preparations contain stabilizers (PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

  • Smaller preparations (20μl) may contain 0.1% BSA as a stabilizer

  • Stable for one year after shipment when stored properly

  • For long-term storage beyond one year, consider storing at -80°C in single-use aliquots

Advanced Research Questions

• How does eIF3J facilitate loading of release factors into the ribosome?

  eIF3J plays a sophisticated role in translation termination by facilitating the binding of release factors to the ribosome:

  • eIF3J significantly enhances peptidyl-tRNA hydrolysis in the presence of release factors eRF1 and eRF3

  • It binds directly to eRF3a or the eRF1-eRF3a complex, but only in the presence of GTP

  • eIF3J cannot interact with eRF1 in solution but can bind to pre-termination complexes

  • It functions in translation termination before GTP hydrolysis by eRF3

  • The N-terminal domain of eRF3a is important for the stimulatory effect of eIF3J on peptide release

  Researchers investigating translation termination mechanisms should consider using toe-printing assays alongside peptidyl-tRNA hydrolysis experiments to precisely determine the step at which eIF3J functions.

• Can eIF3J function in translation termination independent of the eIF3 complex?

  Evidence supports eIF3J's independent function in translation termination:

  • eIF3J significantly stimulates peptide release in pre-termination complexes lacking eIF3

  • The W52A mutation of eIF3J, which weakens interaction with eIF3, does not completely abolish its activity in translation termination

  • This independence is consistent with eIF3J being loosely associated with other eIF3 subunits

  • eIF3J directly binds to ribosomes in pre-termination complexes

  • Both the structured core region and the unstructured N-terminal region of eIF3J are important for its termination activity

  For experiments investigating eIF3J's independent functions, researchers should use purified pre-termination complexes and recombinant eIF3J protein with or without specific domain mutations.

• What are the implications of eIF3 subunits in disease pathology and how can related antibodies be used diagnostically?

  eIF3 subunits have emerging roles in disease mechanisms:

  • Serum anti-EIF3A autoantibody is a potential diagnostic biomarker for hepatocellular carcinoma (HCC)

  • Anti-EIF3A autoantibody combined with other markers (including alpha-fetoprotein) can enhance HCC diagnostic sensitivity from 79.4% to 85%

  • The elevated expression of EIF3A in HCC tissues has been observed in both mouse models and human patients

  • EIF3A exists in tumor-derived exosomes, which may trigger autoantibody production

  Researchers studying the diagnostic potential of eIF3-related markers should develop ELISA assays using autoantibody-specific epitopes. For example, using streptavidin antigen displaying anti-EIF3A autoantibody-specific epitope XC90p2(-CPVRSGFPC-) as capture antigen has shown 79.4% sensitivity and 83.5% specificity for HCC diagnosis .

• What structural and functional domains of eIF3J are critical for experimental design?

  Understanding eIF3J's functional domains is essential for mutagenesis and structure-function studies:

  • The N-terminal deletion variant eIF3J-Δ(1-15) is completely inactive in translation termination

  • The C-terminal deleted variant Δ(243-258) retains approximately 30% of eIF3J activity

  • The W52A mutation reduces activity to approximately 15% of wild-type eIF3J

  • The structured core region contains residues most important for function

  • The unstructured N-terminal region is essential for translational termination activity

  When designing mutations or truncations for functional studies, researchers should consider these differential contributions of various domains to eIF3J activity.

• How can I validate the specificity of eIF3J antibody in complex experimental settings?

  Rigorous validation is essential for reliable experimental outcomes:

  • Perform siRNA/shRNA knockdown or CRISPR knockout of eIF3J followed by Western blot analysis

  • Include positive controls from validated cell lines (HeLa, A549, HEK-293)

  • Compare reactivity with the calculated molecular weight (28 kDa) versus the observed molecular weight (35 kDa)

  • Use immunoprecipitation followed by mass spectrometry to confirm antibody specificity

  • Conduct peptide competition assays to verify epitope specificity

  • For multi-protein complex studies, include appropriate controls for other eIF3 subunits (eIF3A, etc.)

  When studying eIF3J in the context of multi-protein complexes, consider using proximity ligation assays or co-immunoprecipitation to verify protein-protein interactions.

• How should researchers interpret complex data patterns when studying eIF3J's interaction with PABP and other factors?

  eIF3J interacts with multiple factors in translation, requiring nuanced data interpretation:

  • eIF3J and poly(A)-binding protein (PABP) work independently in translation termination and do not suppress each other

  • eRF1, eRF3a, eIF3J, and PABP can form a quadruple complex in the presence of GTP

  • eIF3J and PABP likely interact with different regions of the N domain of eRF3a

  • When interpreting pull-down data, consider that the interaction between eIF3j and eRF3a is nucleotide-dependent and occurs only in the presence of GTP

  • For gradient fractionation experiments, note that eIF3j alone is detected only in the top fractions (1-3)

  When studying these complex interactions, employ multiple complementary techniques (pull-down assays, toe-printing, peptide release assays) and carefully control for nucleotide status in your experimental design.

• What methodological considerations are important when using eIF3J antibodies for immunofluorescence studies?

  Successful immunofluorescence experiments with eIF3J antibodies require careful optimization:

  • Use recommended dilution ranges (1:200-1:800) as starting points for optimization

  • For immunohistochemistry, consider antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

  • When co-staining with other translation factors, select compatible secondary antibodies

  • Since eIF3J associates with the 40S ribosomal subunit, consider counterstaining with markers of translation initiation complexes

  • For super-resolution microscopy, higher antibody concentrations may be required

  • Include appropriate controls for subcellular localization studies, particularly when examining stress granule formation or non-canonical functions of eIF3J

  Document all optimization steps and include representative images of both positive and negative controls in your experimental reports.