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|Baculovirus-Insect Cell lysate that Human USP5 / ISOT transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|A DNA sequence encoding the human USP5 isoform short (P45974-2) (Met 1-Ser 835) was fused with a polyhistidine tag at the C-terminus.|
|The recombinant human USP5 consists of 846 amino acids and predicts a molecular mass of 94.7 kDa. It migrates as an approximately 100 kDa band in SDS-PAGE under reducing conditions.|
|Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
Ubiquitin carboxyl-terminal hydrolase 5, also known as Deubiquitinating enzyme 5, Isopeptidase T, Ubiquitin thiolesterase 5, Ubiquitin-specific-processing protease 5, ISOT and USP5, is a member of the peptidase C19 family. USP5 contains 2 UBA domains and one UBP-type zinc finger. The UBP-type zinc finger domain interacts selectively with an unmodified C-terminus of the proximal ubiquitin. Both UBA domains are involved in polyubiquitin recognition. The UBP-type zinc finger domain crystallizes as a dimer linked by a disulfide bond between the Cys-195 residues of both molecules, but there is no evidence that the full-length USP5 exists as a dimer. USP5 cleaves linear and branched multiubiquitin polymers with a marked preference for branched polymers. USP5 is involved in unanchored 'Lys-48'-linked polyubiquitin disassembly. It binds linear and 'Lys-63'-linked polyubiquitin with a lower affinity. Knock-down of USP5 causes the accumulation of p53/TP53 and an increase in p53/TP53 transcriptional activity because the unanchored polyubiquitin that accumulates is able to compete with ubiquitinated p53/TP53 but not with MDM2 for proteasomal recognition.