GEMIN4 Antibody

Basic Research Questions

• What is GEMIN4 and why is it important in cellular biology?

  GEMIN4 is an essential component of the Survival Motor Neuron (SMN) protein complex, which plays a critical role in the assembly and maturation of Sm-class small nuclear ribonucleoproteins (snRNPs) . The SMN complex is responsible for spliceosomal snRNP assembly in the cytoplasm and regeneration of spliceosomes required for pre-mRNA splicing in the nucleus . GEMIN4 localizes to both the cytoplasm and distinct nuclear bodies called gems, as well as in nucleoli . Studies in mice have demonstrated that GEMIN4 is essential for embryonic development, as null mice die early in embryonic stages . The protein contains nuclear localization signals (NLS) that facilitate nuclear import, and its overexpression can drive SMN and other Gemin proteins from the cytoplasm into the nucleus .

• What are the optimal applications for GEMIN4 antibodies in research?

  GEMIN4 antibodies have been validated for multiple applications with varying optimal dilutions:

  Application	Recommended Dilution	Reference
  Western Blot (WB)	1:200-1:1000
  Immunoprecipitation (IP)	0.5-4.0 μg for 1.0-3.0 mg lysate
  Immunohistochemistry (IHC)	1:100-1:500
  Immunofluorescence (IF)	1:50-1:200
  ELISA	As per manufacturer protocols

  These applications enable researchers to study GEMIN4's expression levels, protein interactions (particularly within the SMN complex), and subcellular localization . For more specialized applications like cellular co-localization studies, immunofluorescence with antibodies against other SMN complex components and nuclear markers is recommended .

• How should GEMIN4 antibodies be validated before use in critical experiments?

  Proper validation of GEMIN4 antibodies should include:

  • Positive control testing using cells known to express GEMIN4 (HEK-293 and HeLa cells are recommended)

  • Negative control analysis using appropriate cell types or knockout/knockdown samples

  • Western blot analysis to confirm detection at the expected molecular weight (120-130 kDa)

  • Cross-reactivity assessment, particularly when working with non-human samples

  • Titration experiments to determine optimal antibody concentration for each specific application

  • Verification of expected subcellular localization pattern (cytoplasmic, nuclear gems, nucleoli) by immunofluorescence

  Remember that near half of commercial antibodies may not function optimally for their recommended applications, making proper validation critical before proceeding with significant experiments .

• What is the expected subcellular localization pattern of GEMIN4?

  GEMIN4 displays a distinctive localization pattern that can be used to validate antibody specificity. In immunofluorescence studies, endogenous GEMIN4 typically:

  • Localizes diffusely throughout the cytoplasm

  • Concentrates in distinct nuclear foci called gems (Gemini bodies), which are often adjacent to Cajal bodies

  • Shows nucleolar staining, unlike other gems proteins

  This tripartite localization pattern makes GEMIN4 unique among SMN complex members. When performing immunofluorescence, co-staining with SMN (which marks gems) and fibrillarin or other nucleolar markers can help confirm proper GEMIN4 detection . Interestingly, when tagged with GFP or myc, GEMIN4 predominantly localizes to the nucleoplasm, which differs from the endogenous pattern .

Intermediate Research Questions

• How do I optimize GEMIN4 antibody dilution for western blotting?

  Optimizing GEMIN4 antibody dilution requires a systematic titration approach:

  1. Begin with a range of dilutions spanning the manufacturer's recommendation (typically 1:200-1:1000)

  2. Use positive control lysates from HEK-293 or HeLa cells, which reliably express GEMIN4

  3. Load varying amounts of protein (5-50 μg) to determine sensitivity thresholds

  4. Assess background signal and specific band intensity at each dilution

  5. Select the dilution that provides the best signal-to-noise ratio while detecting the expected 120-130 kDa band

  For quantitative western blot analysis, verify linear detection range at your chosen antibody concentration. Remember that the highest affinity antibodies can be used at very low concentrations but may cause insufficient staining in situations of antigen excess .

• What epitope retrieval methods work best for GEMIN4 immunohistochemistry?

  For optimal GEMIN4 detection in formalin-fixed, paraffin-embedded (FFPE) tissue sections:

  • Tris-EDTA buffer at pH 9.0 is specifically recommended for GEMIN4 epitope retrieval

  • Heat-induced epitope retrieval (HIER) methods typically provide better results than enzymatic approaches

  • For immunofluorescence on fixed cell preparations, a mild permeabilization with 0.1-0.5% Triton X-100 is usually sufficient

  • When optimizing, compare different retrieval methods side-by-side on serial sections

  Successful epitope retrieval is particularly important for nuclear antigens like GEMIN4, as formaldehyde fixation can mask epitopes through protein cross-linking .

• How can I confirm GEMIN4 antibody specificity in knockdown experiments?

  To validate GEMIN4 antibody specificity through knockdown approaches:

  1. Design siRNAs targeting GEMIN4 (published effective sequences include si-GEMIN4-1)

  2. Transfect siRNAs into appropriate cell lines (HCC1143 and CAL120 have been successfully used)

  3. Confirm knockdown efficiency at mRNA level by qRT-PCR (48 hours post-transfection is recommended)

  4. Perform western blot analysis comparing GEMIN4 detection in knockdown versus control samples

  5. Include functional assays (e.g., cell viability, BrdU incorporation) to confirm biological effects

  In published studies, si-GEMIN4-1 achieved significant knockdown and produced measurable phenotypic effects, including reduced cell viability and decreased BrdU incorporation in breast cancer cell lines .

• What are the best approaches for co-immunoprecipitation of GEMIN4 with other SMN complex components?

  For effective co-immunoprecipitation studies of GEMIN4 and associated proteins:

  1. Use 0.5-4.0 μg of GEMIN4 antibody per 1.0-3.0 mg of total protein lysate

  2. Prepare lysates under non-denaturing conditions to preserve protein-protein interactions

  3. Pre-clear lysates with appropriate control IgG to reduce non-specific binding

  4. Include both positive controls (SMN, Gemin3) and negative controls in your experimental design

  5. Consider reverse IP approaches (precipitating with antibodies against other complex members)

  GEMIN4 directly interacts with Gemin3 and several spliceosome core proteins . For detecting these interactions, using antibodies against the C-terminal region of GEMIN4 may be beneficial, as this region has been implicated in protein-protein interactions .