REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M | Endogenous | 185 | Rabbit IgG |
Western blot analysis of extracts from T-47D cells (serum starved overnight), untreated (-) or treated with Human Neuregulin-1 (hNRG-1) #5218 (100 ng/ml, 15 min; +), using Phospho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb (upper) or HER3/ErbB3 (1B2E) Rabbit mAb #4754 (lower).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded KYSE-450 cell pellets, untreated (left) or treated with Human Epidermal Growth Factor (hEGF) #8916 (right), using Phospho-EGF Receptor (Tyr1068) (D7A5) XP® Rabbit mAb #3777 (upper) or Phospho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb (lower). Note lack of reactivity with Phospho-EGFR.
Learn more about how we get our images.Immunohistochemical analysis of parafin-embedded human lung carcinoma using Phospho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb after retreival with citrate (left) or EDTA (right).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded MCF7 cell pellets, untreated (left) or treated with hNRG-1 #5218 (right), using Phospho-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded HCC827 xenograft, untreated (left) or λ phosphatase-treated (right), using Phospho-HER3/ErbB3 (Tyr1289) (D1B5) 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: 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
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For EDTA: Heat slides in a microwave submersed in 1X EDTA unmasking solution until boiling is initiated; follow with 15 min at a sub-boiling temperature (95°-98°C). No cooling is necessary.
posted February 2010
revised March 2016
Protocol Id: 304
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
Immunohistochemistry (Paraffin) | 1:1600 |
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-HER3/ErbB3 (Tyr1289) (D1B5) Rabbit mAb detects endogenous levels of HER3/ErbB3 proteins only when phosphorylated at Tyr1289. This antibody may cross-react with overexpressed receptor tyrosine kinases.
Human, Mouse
Rat
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr1289 of human HER3/ErbB3 protein.
HER3/ErbB3 is a member of the ErbB receptor protein tyrosine kinase family, but it lacks tyrosine kinase activity. Tyrosine phosphorylation of ErbB3 depends on its association with other ErbB tyrosine kinases. Upon ligand binding, heterodimers form between ErbB3 and other ErbB proteins, and ErbB3 is phosphorylated on tyrosine residues by the activated ErbB kinase (1,2). There are at least 9 potential tyrosine phosphorylation sites in the carboxy-terminal tail of ErbB3. These sites serve as consensus binding sites for signal transducing proteins, including Src family members, Grb2, and the p85 subunit of PI3 kinase, which mediate ErbB downstream signaling (3). Both Tyr1222 and Tyr1289 of ErbB3 reside within a YXXM motif and participate in signaling to PI3K (4).
Investigators have found that ErbB3 is highly expressed in many cancer cells (5) and activation of the ErbB3/PI3K pathway is correlated with malignant phenotypes of adenocarcinomas (6). Research studies have demonstrated that in tumor development, ErbB3 may function as an oncogenic unit together with other ErbB members (e.g. ErbB2 requires ErbB3 to drive breast tumor cell proliferation) (7). Thus, investigators view inhibiting interaction between ErbB3 and ErbB tyrosine kinases as a novel strategy for anti-tumor therapy.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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Product # | Size | Price |
---|---|---|
2842S | 100 µl | $ 303.0 |