Western blot analysis of recombinant monoubiquitin (MonoUb), linear unbranched ubiquitin chain (Ub2-7), K48-linked polyubiquitin (K48-Ub2-7) and K63-linked polyubiquitin (K63-Ub2-7), using K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (upper) and Ubiquitin (P4D1) Mouse mAb #3936 (lower). Ubiquitin chains range from 2 to 7 in length.Learn more about how we get our images.
Western blot analysis of six distinct recombinant polyubiquitin chains using K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (upper) and Ubiquitin (P4D1) Mouse mAb #3936 (lower).Learn more about how we get our images.
Western blot analysis of H1703 cells, untreated or treated with MG132 as indicated, using K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb.Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
K48-linkage Specific Polyubiquitin (D9D5C6) Rabbit mAb detects polyubiquitin chains formed by Lys48 residue linkage. This antibody does not react with monoubiquitin or polyubiquitin chains formed by specific linkage to a different lysine residue.
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding the Lys48 branch of the human diubiquitin chain.
Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25A, and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7).
Substrate proteins are linked to ubiquitin using seven distinct ubiquitin lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63). Formation of a polyubiquitin chain occurs when a lysine residue of ubiquitin is linked to the carboxy-terminal glycine of another ubiquitin. Proteins polyubiquinated at specific lysine residues display a tendency to be targeted for different processes; K48-linked polyubiquitin chains mainly target proteins for proteasomal degradation while K63-linked polyubiquitin regulates protein function, subcellular localization, or protein-protein interactions (8).
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