Details
Description
This kit is designed for enrichment of polyubiquitinated cellular proteins using GST tagged recombinant proteins that bind lysine 48 and lysine 63 polyubiquitin chains. It can be used to enrich polyubiquitinated proteins in whole cell or tissue lysates. Bound proteins can be precipitated using glutathione resin and eluted by a buffer containing 10 mM glutathione, and then assayed by immunoblotting or mass spectrometry.
Components
• GST-S5a (UIM)/ GST-TAB2 (NZF) Mix (2 mg/ml each)..............1 ml
• GST (4 mg/ml)...........................................................................0.5 ml
• Iodoacetamide (IAA)..................................................................25 mg
• Glutathione (reduced)..............................................................100 mg
• Glutathione Agarose Resin (50% slurry)..................................1.5 ml
Images:
(Click image to enlarge)
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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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