K48 Polyubiquitin Chain Capture Kit

This kit is designed for enrichment of cellular proteins conjugated with lysine 48-linked polyubiquitin chains using GST-S5a (UIM). Polyubiquitinated proteins bound on GST-S5a (UIM) can be precipitated using gluta-thione resin and eluted by a buffer containing 10 mM glutathione. Polyubiquitinated proteins can be as-sayed by immunoblotting or mass spectrometry.

Additional Information

Product Name: K48 Polyubiquitin Chain Capture Kit
Also Known As: N/A
Catalog No.: J4430
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 cellular proteins conjugated with lysine 48-linked polyubiquitin chains using GST-S5a (UIM). Polyubiquitinated proteins bound on GST-S5a (UIM) can be precipitated using gluta-thione resin and eluted by a buffer containing 10 mM glutathione. Polyubiquitinated proteins can be as-sayed by immunoblotting or mass spectrometry.

Components

• GST-S5a (UIM) (4 mg/ml).........................................0.5 ml
• GST (4 mg/ml)..........................................................0.5 ml
• Iodoacetamine (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.