Western blot analysis of extracts from HCT 116 cells, G0 cells that were serum starved and contact inhibited (36 hr) or mitotic cells treated with thymidine (2 mM, 18 hr) followed by Nocodazole #2190 (50 ng/ml, 20 hr), using Phospho-Thr-Pro-Pro Motif [pTPP] (D61C3) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (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
Phospho-Thr-Pro-Pro Motif [pTPP] (D61C3) Rabbit mAb recognizes endogenous levels of proteins only when phosphorylated within the TPP motif. It does not cross react with phospho-serine or phospho-threonine in other contexts.Species Reactivity:
All Species Expected
Monoclonal antibody is produced by immunizing animals with synthetic peptide library containing the phospho-Thr-Pro-Pro motif.
The phospho-Thr-Pro-Pro motif is a subgroup of the phospho-Thr-Pro motif that is phosphorylated by proline-directed protein kinases, including MAP kinases and cyclin dependent kinases (CDKs). Proline-directed phosphorylation is one of the major regulatory phosphorylation events in cell proliferation, cell differentiation, and a number of other essential cellular processes (1-6). This motif was identified in both phospho-proteomic and motif X analyses as a significant phospho-thr motif (7,8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at email@example.com.