Western blot analysis of extracts from AR42J cells untreated (-) or treated with 1 μM thapsigargin (Tg) for 20 minutes (+), using Phospho-PERK (Thr980) (16F8) Rabbit mAb.Learn more about how we get our images.
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
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.
Phospho-PERK (Thr980) (16F8) Rabbit mAb detects endogenous levels of PERK phosphorylated at Thr980.
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr980 of mouse PERK.
Protein kinase-like endoplasmic reticulum kinase (PERK) is an eIF2α kinase and transmembrane protein resident in the endoplasmic reticulum (ER) membrane that couples ER stress signals to translation inhibition (1-3). ER stress increases the activity of PERK, which then phosphorylates eIF2α to promote reduced translation. Research studies have demonstrated that PERK-deficient mice have defects in pancreatic β cells several weeks after birth, suggesting a role for PERK-mediated translational control in protecting secretory cells from ER stress (4). PERK activation during ER stress correlates with autophosphorylation of its cytoplasmic kinase domain (1-3). Phosphorylation of PERK at Thr980 serves as a marker for its activation status.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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