The protein interactions between the CIDE domains and various mutants were analyzed by native (non-denaturing) PAGE using a PhastSystem (GE Healthcare) with pre-made 8–25% acrylamide gradient gels (GE Healthcare). The separated and purified proteins were pre-incubated at room temperature for 1 h before loading onto the gel, and Coomassie Brilliant Blue was used to stain and detect the shifted bands.
Acrylamide gradient gels
Manufactured by GE Healthcare
8–25% acrylamide gradient gels are a type of laboratory equipment used for protein electrophoresis. These gels are designed to separate proteins based on their molecular weight. The gradient in the gel allows for efficient separation of a wide range of protein sizes in a single run.
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9 protocols using acrylamide gradient gels
1
CIDE Domain Protein Interactions
2
Monitoring Oligomerization Changes by Native PAGE
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Changes in oligomerization in response to mutations and complex formation were monitored by native (non-denaturing) PAGE on a PhastSystem system (GE Healthcare) with pre-made 8–25% acrylamide gradient gels (GE Healthcare). Separately purified proteins were pre-incubated at 20 °C for 1 h before loading the gel. Coomassie Brilliant Blue was used for staining and detection of the shifted bands.
3
Native PAGE Analysis of RAIDD:PIDD Complex
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Formation of the complex was assayed by native (non-denaturing) PAGE conducted on a PhastSystem (GE Healthcare) with pre-made 8–25% acrylamide gradient gels (GE Healthcare). Coomassie brilliant blue was used for staining and detection of the bands. RAIDD DD:PIDD DD and RAIDD:PIDD DD complexes were prepared in Tris buffer (20 mM Tris pH 8.0 and 50 mM NaCl). This complex was then pre-incubated with RDH3 or PDH3 for 1 hour, after which the mixture was subjected to the gel. The percentage of the complex was evaluated based on the appearance of newly formed bands. The amount of the complex formed without peptides was considered to be 100%.
4
CARD9-BCL10 Interaction Characterization
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Formation of the complex between CARD9 and BCL10 CARDs was assessed using native (non-denaturing) PAGE on an Amersham™ PhastSystem (GE Healthcare Life Sciences) with pre-made 8–25% acrylamide gradient gels (GE Healthcare Life Sciences). Coomassie Brilliant Blue dye was used for the detection of protein band patterns. Separately purified CARD9 and BCL10 CARDs were pre-incubated together for 1 h at room temperature and the mixture was applied to the gel. Complex assembly was evaluated based on the appearance of newly formed bands and disappearance of existing bands.
5
IRGB10-GBP Complex Formation Assay
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The formation of complexes between IRGB10 and GBPs was assayed by native (non-denaturing) PAGE conducted on a PhastSystem (GE Healthcare) with pre-cast 8–25% acrylamide gradient gels (GE Healthcare). Coomassie brilliant blue was used for the staining and detection of bands. IRGB10 was mixed with GBP5 or GBP2 in the absence and presence of GTP and MgCl2, and incubated for 1 h at 4 °C, after which the mixture was subjected to electrophoresis. Complex formation was evaluated based on the appearance of newly formed bands or the disappearance of bands that were detected in single control protein bands.
6
Oligomeric Forms of CIDE Domains
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Oligomeric forms of each CIDE domains were monitored by native (non-denaturing) PAGE on a PhastSystem (GE Healthcare) with pre-made 8–25% acrylamide gradient gels (GE Healthcare). Separately purified proteins were directly loaded onto the gel. Coomassie Brilliant Blue was used for staining and detection of the shifted bands. The uncropped scan of gel is provided at Supplementary Fig. 1 .
7
Native PAGE Analysis of TRAIP Complexes
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The oligomeric states of TRAIP CC and TRAIP RING-CC, as well as the complex formation of various TRAIP proteins with TRAF, were monitored by Native (non-denaturing) PAGE using a Phast System (GE Healthcare) with pre-made 8–25% acrylamide gradient gels (GE Healthcare). Purified protein samples obtained by affinity and size-exclusion chromatography were loaded onto the gel. Coomassie Brilliant Blue was used for staining.
8
Monitoring Oligomerization via Native PAGE
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Changes in oligomerization in response to mutations were monitored by native (non-denaturing) PAGE using a Phast System (GE Healthcare) with pre-made 8–25% acrylamide gradient gels (GE Healthcare). Separately purified proteins were pre-incubated at room temperature for 1 hour before loading the gel. Coomassie Brilliant Blue was used for staining and detection of shifted bands.
9
Monitoring IRGB10 Self-Oligomerization
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Self-oligomerization of IRGB10 due to GTPase activity was monitored by native-PAGE using a Phast system (GE Healthcare). Pre-made 8%–25% acrylamide gradient gels (GE Healthcare) were used for this experiment. The shifted bands on the gel were stained with Coomassie Brilliant Blue. Purified nucleotide-free IRGB10 was mixed and incubated with different concentrations of GTP and MgCl2 mixtures at 37°C for 30 min, before loading the mixture onto native gels.
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