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Tetra-ubiquitin (K48-linked)

 
BML-UW8645-0025 25 µg 244.00 USD
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Biochemically useful quantities of polyubiquitinylated target proteins are a pre-requisite for dissecting the mechanistic co-ordination of chain recognition, substrate unfolding, and peptide bond hydrolysis by the 26S proteasome. Di-ubiquitin (Ub2) is made by incubating E2-25k (Prod. No. BML-UW9020) with equal concentrations of ubiquitin capped at the future proximal chain terminus (Asp77-ubiquitin) and ubiquitin capped at the future distal chain terminus (K48C-ubiquitin). The proximal or distal terminus of the resulting dimer is deblocked by treatment with Ubiquitin hydrolase L3 (Prod. No. BML-UW9745) or ethyleneimine, respectively. In the second cycle of synthesis, tetra-ubiquitin (Ub4) is made by incubating the two single-deblocked dimers with E2-25k. Following purification by anion and cation exchange chromatography, the distal Cys48 is blocked by reaction with iodoacetamide, which is then removed by dialysis. Ub4 retains Asp77 at its proximal terminus.

Product Specification

Alternative Name:Ub4
 
MW:~32kDa
 
Source:Produced in E. coli and biochemically synthesized in vitro.
 
UniProt ID:P0CG47 (UBB), P0CG48 (UBC)
 
Formulation:Liquid, containing 50mM TRIS pH7.5, 1mM EDTA and 5mM DTT.
 
Purity:≥85% (SDS-PAGE)
 
Purity Detail:Ubiquitin mutants [K48C] and [D77] were expressed in BL21 (λDE3) . Proteins were purified by cation exchange chromatography. Di-ubiquitin was synthesised by linking [K48C]ubiquitin and [D77]ubiquitin in the presence of ubiquitin conjugating enzyme E2-25K (UbcH1) (Prod. No. BML-UW9020) and human ubiquitin activating enzyme E1 (Prod. No. BML-UW9410). The product was purified by ion exchange chromatography. Tetra-ubiquitin is made by incubating the two single-deblocked lysines blocked by reaction with iodacetamide. Tetra-ubiquitin retains Asp. Purity was confirmed by SDS-PAGE and protein concentration estimated by absorbance at 280nm.
 
Application Notes:Useful for studying the mechanism of chain recognition, substrate unfolding, and peptide bond hydrolysis by the 26S proteasome.
 
Shipping:Shipped on Blue Ice
 
Long Term Storage:-20°C
 
Handling:Avoid freeze/thaw cycles.
 
Technical Info/Product Notes:PLEASE NOTE: Tetra-ubiquitin may undergo complex formation to give higher molecular weight multimers upon heating and, consequently, the duration of any heating steps during use should be minimised. The exact nature of the multimeric complexes is not known at this time.
 
BML-UW8645 SDS-PAGE
SDS-PAGE Analysis. Lane 1: MW Marker, Lane 2: 1 µg Tetra-Ubiquitin
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BML-UW8645 SDS-PAGE

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