Details
Description
This kit is designed for enrichment of cellular proteins conjugated with linear polyubiquitin chains using GST-NEMO (UBAN). Polyubiquitinated proteins bound on GST-NEMO (UBAN) can be precipitated using glutathione resin and eluted by a buffer containing 10 mM glutathione. Polyubiquitinated proteins can be assayed by immunoblotting or mass spectrometry.
Components
• GST-NEMO (UBAN) (4 mg/ml)..................................0.5 ml
• GST (4 mg/ml)...........................................................0.5 ml
• Iodoacetamide (IAA)..................................................25 mg
• Glutathione (reduced)...............................................100 mg
• Glutathione Resin (50% slurry).................................1.5 ml
Images:
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Schematic of polyUb chains enrichment
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Lane 1: GST
Lane 2: GST-S5a (UIM)
Lane 3: GST-TAB2 (NZF)
Lane 4: GST-S5a (UIM) + GST-TAB2 (NZF)
Lane 5: GST-NEMO (UBAN)
According to procedures described above, 5 mg whole cell lysates from HeLa cells were mixed with 0.2 mg GST (lane 1), GST-S5a (UIM) (lane 2), GST-TAB2 (NZF) (lane 3), GST-S5a (UIM) + GST-TAB2 (NZF) (lane 4), or GST-NEMO (UBAN) (lane 5). 10% eluates were separated by SDS-PAGE, followed by immunoblotting of Ub, K48-specific Ub chains and GST.
* No immunoreactivity was detected in GST-NEMO (UBAN) pulldown assays, presumably because there is little linear polyubiquitin chains in cells.
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