商品编号


J4310



品牌


UBPBio



公司


Ubiquitin-Proteasome Biotechnologies, LLC



公司分类


Proteasomes / Activators / Subunits



Size

1 Kit



商品信息

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-S



Also Known As:

N/A



Catalog No.:

J4310



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
,
MSD
S



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 50 mg or 5 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)...................................................0.6 ml
? GST (4 mg/ml)..........................................................0.5 ml ? 6xHis-UIM (4 mg/ml).............................................. 0.2 ml
? 20X Lysis Buffer........................................................1.2 ml ? NaCl (1M)...............................................................1.25 ml
? ATP (500mM)............................................................0.1 ml ? SUC-LLVY-AMC (50 mM)..........................................10 ?l
? Glutathione Agarose Resin (75% slurry)...........1.0 ml ? Nickel XPure Agarose Resin (50% slurry)..........500 ?l

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