REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H | Endogenous | 120 | Rabbit IgG |
Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with pervanadate (1 mM; +), using Phospho-c-Cbl (Tyr700) (D16D7) Rabbit mAb (upper) or c-Cbl (D4E10) Rabbit mAb #8447 (lower).
Learn more about how we get our images.Western blot analysis of extracts from Jurkat cells, untreated (-) or treated with pervanadate (1 mM; +), using Phospho-c-Cbl (Tyr700) (D16D7) Rabbit mAb (left). The phospho-specificity of the antibody was verified by preincubating the antibody with c-Cbl (Tyr700) phosphopeptide (center) or with c-Cbl (Tyr700) nonphosphopeptide prior to incubation with the membrane (right).
Learn more about how we get our images.Immunoprecipitation (IP) of phosphorylated c-Cbl from Jurkat cell extracts, untreated (-) or treated with pervanadate (1 mM; +), using Phospho-c-Cbl (Tyr700) (D16D7) Rabbit mAb (lanes 4 and 6). Western blot detection was performed using the same antibody. Lanes 1 and 2 are 10% input. Lanes 3 and 5 are IPs performed with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900.
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: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 410
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
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-c-Cbl (Tyr700) (D16D7) Rabbit mAb recognizes endogenous levels of c-Cbl protein only when phosphorylated at Tyr700.
Human
Mouse
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr700 of human c-Cbl protein.
The c-Cbl proto-oncogene is a ubiquitously expressed cytoplasmic adaptor protein that is especially predominant in hematopoietic cells (1,2). c-Cbl is rapidly tyrosine-phosphorylated in response to stimulation of a variety of cell-surface receptors and becomes associated with a number of intracellular signaling molecules such as protein tyrosine kinases, phosphatidylinositol-3 kinase, Crk, and 14-3-3 proteins (3,4). c-Cbl possesses a highly conserved amino-terminal phosphotyrosine binding domain (TKB) and a C3HC4 RING finger motif. The TKB recognizes phosphorylated tyrosines on activated receptor tyrosine kinases (RTKs) as well as other nonreceptor tyrosine kinases. The RING finger motif recruits ubiquitin-conjugating enzymes. These two domains are primarily responsible for the ubiquitin ligase activity of c-Cbl and downregulation of RTKs (3). Research studies have indicated that in human cancer tissues, c-Cbl is frequently tyrosine-phosphorylated in a tumor-specific manner (5). Phosphorylation of Tyr731 of c-Cbl provides a docking site for downstream signaling components such as p85 and Fyn (6).
It has been demonstrated that c-Cbl is phosphorylated at Tyr700 by Fyn, Yes, and Syk (4) and that Vav, a hematopoietic-restricted Rac guanine nucleotide exchange factor, undergoes c-Cbl-dependent ubiquitination upon recruitment to phospho-Tyr700 (5).
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8869T | 20 µl | $ 122.0 |
8869S | 100 µl | $ 303.0 |