Western blot analysis of extracts from HeLa cells, untreated (-) or treated with tunicamycin (2 μg/ml, 8 hr; +), using XBP-1s (D2C1F) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human XBP-1s (hXBP-1s-Myc/DDK; +), using XBP-1s (D2C1F) Rabbit mAb (upper), DYKDDDDK Tag (9A3) Mouse mAb #8146 (middle) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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 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 June 2020
Protocol Id: 263
XBP-1s (D2C1F) Rabbit mAb recognizes endogenous levels of the total spliced isoform of XBP1 protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu64 of human XBP1 protein.
Following protein synthesis, secretory, intra-organellar, and transmembrane proteins translocate into the endoplasmic reticulum (ER) where they are post-translationally modified and properly folded. The accumulation of unfolded proteins within the ER triggers an adaptive mechanism known as the unfolded protein response (UPR) that counteracts compromised protein folding (1). The transmembrane serine/threonine kinase IRE1, originally identified in Saccharomyces cerevisiae, is a proximal sensor for the UPR that transmits the unfolded protein signal across the ER membrane (2-4). The human homolog IRE1α was later identified and is ubiquitously expressed in human tissues (5). Upon activation of the unfolded protein response, IRE1α splices X-box binding protein 1 (XBP-1) mRNA through an unconventional mechanism using its endoribonuclease activity (6). This reaction converts XBP-1 from an unspliced XBP-1u isoform to the spliced XBP-1s isoform, which is a potent transcriptional activator that induces expression of many UPR responsive genes (6).
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