1. Ubiquitinylation of specific endogenous HeLa lysate proteins, followed by their immediate detection/analysis using antibodies to the protein/s of interest, indicating a particular protein is a substrate for the ubiquitin-proteasome pathway. 2. Ubiquitinylation of proteins of interest contained in exogenously added expression culture/cell extracts or tissue lysates/extracts, followed by their immediate detection/analysis or isolation/purification for use in subsequent experiments. 3. Modification of proteins using ubiquitin derivatives or mutants for improved detection/analysis or investigation of alternative (non-proteasomal) ubiquitin related pathways in subsequent experiments. For example, biotinylated-ubiquitin (Prod. No. BML-UW8705), for sensitive detection
For the generation of ubiquitin conjugated proteins. HeLa S100 lysate facilitates controlled ubiquitin conjugation of substrate proteins (exogenous or endogenous) of interest through the ubiquitin cascade. Conjugate formation can be detected and monitored by Western blotting using the highly sensitive ubiquitin-conjugate specific antibody supplied and/or antibodies for specific target proteins. Modified proteins can be subjected to further purification prior to their use in subsequent experiments if required. Suggested uses: Generation of ubiquitin conjugated proteins. Exogenous or endogenous HeLa lysate proteins (tagged/radio-labeled/immuno-detectable) can be ubiquitinylated followed by immediate detection/analysis. Subsequent analysis could include proteasomal degradation, ubiquitin modification site mapping (by mass spectrometry), and the effect of ubiquitin modification on enzyme interactions, activity and function, Ubiquitinylation of proteins of interest from cell or tissue extracts, Modification of proteins using ubiquitin derivatives or ubiquitin mutants for improved detection, analysis or investigation of alternative (non-proteasomal) ubiquitin signaling pathways.
Ordering information: Quantity: Provides sufficient material for 20×50 μl reactions. Contents: HeLa S100 fraction (Prod. No. BML-SW8750)1 mg (200 µl); 5 mg/ml 10x Ubiquitin solution (UB) (Prod. No. BML-KW8795)100 µl; 1 mg/ml10x ATP-(Energy) regeneration solutions (ATP-ERS) (Prod. No. BML-EW9810)100 µl10x Ubiquitinylation buffer (Prod. No. BML-KW9885a)100 µlUbiquitin aldehyde (inhibitor) (Prod. No. BML-UW8450)50 µl; 1mg/mlUbiquitin-conjugate specific antibody solution (FK2) (Prod. No. BML-KW8810)10 µl, Mono- and polyubiquitinylated proteins, multi-ubiquitin chains, mouse mAb (clone FK2)