Western blot analysis of extracts from various cell lines using 4-1BBL/CD137L/TNFSF9 (D6V6K) Rabbit mAb (upper) and β-actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
Western blot analysis of extracts from THP-1 cells, untreated (-) or treated with Lipopolysaccharide (LPS) #14011 (100 ng/ml, 7 hr; +), using 4-1BBL/CD137L/TNFSF9 (D6V6K) Rabbit mAb (upper) and β-actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
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
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.
4-1BBL/CD137L/TNFSF9 (D6V6K) Rabbit mAb recognizes endogenous levels of total 4-1BBL/CD137L/TNFSF9 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala246 of human 4-1BBL/CD137L/TNFSF9 protein.
TNFRSF9 is a member of the tumor necrosis factor receptor superfamily (1, 2). It is also called 4-1BB or CD137 (1, 2). 4-1BB/CD137/TNFRSF9 is expressed in activated CD4+ and CD8+ T cells, natural killer cells and dendritic cells (2-5). The ligand 4-1BBL/CD137L/TNFSF9 on antigen presenting cells binds to 4-1BB/CD137/TNFRSF9 and costimulates the activation of T cells (5). The binding of agonistic antibodies to 4-1BB/CD137/TNFRSF9 also leads to costimulation for T cell activation (5). Studies have shown the effectiveness of targeting 4-1BB/CD137/TNFRSF9 by its agonistic antibodies in cancer immunotherapy (6).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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