Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Tapasin protein (hTapasin-Myc/DDK; +), using Tapasin Antibody (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from HeLa and B16-F10 cells, untreated (-) or treated with IFN-γ (100 ng/ml, 16 hr; +), using Tapasin Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. 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
Tapasin Antibody recognizes endogenous levels of total Tapasin protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Tapasin protein. Antibodies are purified by peptide affinity chromatography.
TAP-associated glycoprotein (Tapasin/TAPBP) is a type-I transmembrane ER glycoprotein encoded by an MHC-linked gene. Tapasin plays a critical role in the MHC class I-restricted antigen processing and presentation pathway. Research studies have demonstrated that Tapasin is an IFNγ-inducible component of the TAP-1/2 peptide transporter complex and, within the ER lumen, facilitates the optimization of the peptide repertoire that is loaded onto MHC class I molecules (1-5). Downregulation of Tapasin and MHC class I expression has been observed in human tumors and serves as a prominent mechanism of tumor immune escape (6-9).
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