Western blot analysis of extracts from various cell lines using Pellino-1 (D2Z4F) Rabbit mAb.
Western blot analysis of 293T cells, mock tranfected (-) or transfected with a construct expressing full-length human Myc/DDK-tagged Pellino-1 (hPellino-1-Myc/DDK; +), using Pellino-1 (D2Z4F) 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 nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
Pellino-1 (D2Z4F) Rabbit mAb recognizes endogenous levels of total Pellino-1 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala403 of human Pellino-1 protein.
Pellino-1 (PELI1) is an E3 ubiquitin ligase that regulates activation of various immune pathways in different contexts and cell types (1). Pellino-1 controls NFκB activation downstream of the interleukin-1 receptor and TLRs through association with and ubiquitination of IRAK1 (1-4). Pellino-1 was also demonstrated to regulate the NFκB pathway in T cells by targeting c-Rel for degradation, thereby enabling suppression by regulatory T cells, maintaining peripheral tolerance, and preventing autoimmunity in mice (5). Finally, in experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis, Pellino-1 contributes to inflammation by promoting degradation of Traf3 leading to induction of chemokines and proinflammatory cytokines (6).
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