Western blot analysis of extracts from cells expressing various activated tyrosine kinase proteins, using Phospho-c-Abl (Tyr245) (73E5) Rabbit mAb (upper) or Phospho-Tyrosine mAb (P-Tyr-100) #9411 (lower).
Western blot analysis of CML-T1 and BV173 cell lysates, using Phospho-c-Abl (Tyr245) (73E5) Rabbit mAb. The phospho-specificity of the antibody was verified by treating the membrane with (+) (right) or without (-) (left) calf intestinal phosphatase (CIP) after Western transfer.
|MW (kDa)||135 (c-Abl); 210 (Bcr-Abl)|
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 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-c-Abl (Tyr245) (73E5) Rabbit mAb detects endogenous levels of c-Abl only when phosphorylated at tyrosine 245. The antibody does not cross-react with other activated protein tyrosine kinases.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr245 of human c-Abl.
The c-Abl proto-oncogene encodes a nonreceptor protein tyrosine kinase that is ubiquitously expressed and highly conserved in metazoan evolution. c-Abl protein is distributed in both the nucleus and the cytoplasm of cells. It is implicated in regulating cell proliferation, differentiation, apoptosis, cell adhesion, and stress responses (1-3). c-Abl kinase activity is increased in vivo by diverse physiological stimuli including integrin activation; PDGF stimulation; and binding to c-Jun, Nck, and RFX1 (2,4). The in vivo mechanism for regulation of c-Abl kinase activity is not completely understood. Tyr245 is located in the linker region between the SH2 and catalytic domains. This positioning is conserved among Abl family members. Phosphorylation at Tyr245 is involved in the activation of c-Abl kinase (5). In addition, phosphorylation at Tyr412, which is located in the kinase activation loop of c-Abl, is required for kinase activity (6).
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.