Primary antibodies against the following proteins were used: rabbit anti-RNP-8 (Kim et al. 2009 (link)); mouse anti-NPC (Mab414, Covance), anti-tubulin (T5168, Sigma), and anti-GLD-2 (Millonigg et al. 2014 (link)); and rat anti-GLD-3 (Eckmann et al. 2002 (link)). The mouse anti-GLD-2 (Millonigg et al. 2014 (link)) recognized the C terminus of the protein. The rabbit anti-GLD-2 antibody used in the immunoprecipitation experiments was generated by immunizing New Zealand white rabbits with an epitope that covered amino acid 959–1113 of GLD-2, fused to a GST-affinity tag. The fusion protein had been expressed in BL21 bacteria and purified via a GST column to homogeneity. An analogously prepared MBP-fusion protein had been coupled to Hi-TRAP NHS columns (GE Healthcare) and used for affinity purification of the immune serum rb184.
Protocol detail
Antibody Generation and Characterization
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Affinity purification
Amino acid
Anti antibody
Antibodies
Bacteria
Epitope
Immune serum
Immunoprecipitation
Mouse
New zealand white rabbits
Protein
Protein c
Rabbit
Tubulin
Corresponding Organization :
Other organizations : Martin Luther University Halle-Wittenberg
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Variable analysis
independent variables
- Rabbit anti-RNP-8
- Mouse anti-NPC
- Anti-tubulin (T5168, Sigma)
- Anti-GLD-2 (Millonigg et al. 2014)
- Rat anti-GLD-3 (Eckmann et al. 2002)
- Not explicitly mentioned
- Mouse anti-GLD-2 (Millonigg et al. 2014) recognized the C terminus of the protein
- Rabbit anti-GLD-2 antibody used in the immunoprecipitation experiments was generated by immunizing New Zealand white rabbits with an epitope that covered amino acid 959–1113 of GLD-2, fused to a GST-affinity tag
- The fusion protein had been expressed in BL21 bacteria and purified via a GST column to homogeneity
- An analogously prepared MBP-fusion protein had been coupled to Hi-TRAP NHS columns (GE Healthcare) and used for affinity purification of the immune serum rb184
- Not explicitly mentioned
- Not explicitly mentioned
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