Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human UBE1 protein (hUBE1-Myc/DDK; +), Myc/DDK-tagged full-length human UBE1L2 protein (hUBE1L2-Myc/DDK; +) and Myc/DDK-tagged full-length human UBE1L protein (hUBE1L-Myc/DDK; +), using UBE1L/UBA7 (D6U4S) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).
Western blot analaysis of extracts from various cell lines using UBE1L/UBA7 (D6U4S) Rabbit mAb.
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.
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
UBE1L/UBA7 (D6U4S) Rabbit mAb recognizes endogenous levels of total UBE1L/UBA7 protein. This antibody does not cross-react with either UBE1/UBA1 or UBE1L2/UBA6 proteins.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human UBE1L/UBA7 protein.
Interferon-stimulated 15 kDa protein (ISG15), also known as ubiquitin cross-reactive protein (UCRP), is a member of the ubiquitin-like protein family and functions in various biological pathways from pregnancy to innate immune responses (1). Expression of ISG15 is stimulated by cellular exposure to type 1 interferons α and β, in addition to infection with viruses such as influenza B (2,3). After exposure to type I interferons, both lymphocytes and monocytes, in addition to some fibroblasts and epithelial cells, release ISG15 into culture medium (1,4). ISG15 has been shown to function as a cytokine, stimulating interferon γ secretion by monocytes and macrophages, proliferation of natural killer cells, and chemotactic responses in neutrophils (4,5). ISG15 has also been shown to function intracellularly, being covalently conjugated to other proteins by E1 (Ube1L), E2 (UbcH8) and E3 ligases via a multi-step process analogous to ubiquitination (6,7). ISG15 is removed from proteins by the ubiquitin processing protease Ubp43 (8). ISG15-protein conjugation (ISGylation) is induced by type 1 interferons, and target proteins include the serine protease inhibitor Serpin 2A, PLCγ1, ERK1/2, Jak1 and Stat1 (9,10). Unlike ubiquitination, ISGylation does not target proteins for degradation, rather ISGylation increases Jak1 and Stat1 activity, enhancing the cellular response to interferons (11).
Ubiquitin-activating enzyme E1-like protein/Ubiquitin-activating enzyme 7 (UBE1L/UBA7) is the activating enzyme for ISG15. Research studies have suggested that loss of UBE1L/UBA7 expression contributes to the development of lung cancer due to compromised inhibition of cyclin D1 expression (12-15). UBE1L/UBA7 has also been implicated in the pathogenesis of acute promyelocytic leukemia through a mechanism in which UBE1L/UBA7 drives ISG15ylation of the oncogenic PML-RARα fusion protein to promote its degradation (16,17).
Explore pathways + proteins related to this product.
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