Polyubiquitin Chain Capture Kit

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 im-munoblotting or mass spectrometry.

Additional Information

Product Name: Polyubiquitin Chain Capture Kit
Also Known As: N/A
Catalog No.: J4460
Size: 1 Kit
Molecular Weight: N/A
Species: N/A
Source: N/A
Stock: Enzyme stocked in 20 mM Tris, 150 mM NaCl, 2 mM βME, 10% Glycerol
Concentration: See tube label
Quality Assurance: Enzyme >95% by SDS-PAGE
Storage: Store at -80°C; avoid multiple freeze-thaw cycles
PDF Data Sheet: PDF datasheet, MSDS
NCBI RefSeq: N/A
Image(s): (Click image to enlarge)

Schematic of polyUb chains enrichment

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.
Shipping Method: Dry ice shipping
References: N/A

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 (75% slurry)..................................1.0 ml

Images:
(Click image to enlarge)

Schematic of polyUb chains enrichment

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.