Rapid 26S Proteasome Purification Kit-L

The Rapid 26S Proteasome Purification Kit is designed to rapidly purify the endogenous 26S proteasome from cell or tissue extracts within 8 hours according to a published method (reference 1) with modifications. This approach uses the N-terminal ubiquitin-like (Ubl) domain of human RAD23B as an affinity bait, which allows the rapid and gentle isolation of endogenous 26S proteasomes. The bound 26S proteasome is subsequently eluted using the C-terminal two ubiquitin-interacting motifs (UIM) of human S5a.

Additional Information

Product Name: Rapid 26S Proteasome Purification Kit-L
Also Known As: N/A
Catalog No.: J4320
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; Substrate stocked in DMSO
Concentration: See tube label
Quality Assurance: Enzyme >95% by SDS-PAGE; Substrate > 98% by HPLC and NMR
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 26S proteasome enrichment
Shipping Method: Dry ice shipping
References: 1. Besche HC, Haas W, Gygi SP, Goldberg AL. Biochemistry. 2009; 48:2538-49.
2. Elsasser S, Schmidt M, Finley D. Methods Enzymol. 2005; 398:353-63.
3. Kisselev AF, Goldberg AL. Methods Enzymol. 2005; 398:364-78.

Details

This kit is sufficient for purification of the 26S proteasome from 300 mg, 6 X 50 mg or 30 X 10 mg cell or tissue extracts at a concentration of 5 mg/ml. Usually, 1-2% of total cellular proteins are the 26S proteasome.

Description

The Rapid 26S Proteasome Purification Kit is designed to rapidly purify the endogenous 26S proteasome from cell or tissue extracts within 8 hours according to a published method (reference 1) with modifications. This approach uses the N-terminal ubiquitin-like (Ubl) domain of human RAD23B as an affinity bait, which allows the rapid and gentle isolation of endogenous 26S proteasomes. The bound 26S proteasome is subsequently eluted using the C-terminal two ubiquitin-interacting motifs (UIM) of human S5a.

Components

• GST-Ubl (4 mg/ml)...................................................2 X 1.2 ml
• GST (4 mg/ml).........................................................2 X 1.2 ml
• 6xHis-UIM (4 mg/ml)....................................................1 X 1 ml
• 20X Lysis Buffer........................................................3 X 1.2 ml
• NaCl (1M)...............................................................2 X 1.25 ml
• ATP (500mM)................................................................. 0.5 ml
• SUC-LLVY-AMC (50 mM)..................................................30 µl
• Glutathione Agarose Resin (50% slurry)...........2 X 1.5 ml
• Nickel XPure Agarose Resin (50% slurry)..........2 X 500 µl

Images
(Click image to enlarge)


A. Coomassie stained SDS-PAGE of 5 μg purified GST-Ubl.
B. Coomassie stained SDS-PAGE of 5 μg purified 6xHis-UIM.
C. Coomassie stained SDS-PAGE of 3 μg purified human 26S proteasomes.
D. Coomassie stained native-PAGE of 3 μg purified human 26S proteasomes.
E. Solution peptidase assay of the purified human 26S proteasome. 150 μl reaction mixtures contained 10 nM purified human 26S proteasome in 50 μM SUC-LLVY-AMC in 20 mM Tris, pH 7.1 at 37°C, 2 mM μME. The released AMC fluorescence was measured using a plate reader with excitation and emission filters at 360/40 and 460/30 nm, respectively. This assay was used to monitor the chymotrypsin-like activity of the 26S proteasome. (Click image to enlarge)

Schematic of 26S proteasome enrichment