REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M R Mk | Endogenous | 15-20 | Rabbit IgG |
Western blot analysis of extracts from various cell lines using 4E-BP1 (53H11) Rabbit mAb.
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded human breast carcinoma using 4E-BP1 (53H11) Rabbit mAb in the presence of control peptide (left) or 4E-BP1 blocking peptide #1053 (right).
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using 4E-BP1 (53H11) Rabbit mAb.
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded human lung carcinoma using 4E-BP1 (53H11) Rabbit mAb.
Learn more about how we get our imagesImmunohistochemical analysis of paraffin-embedded mouse spleen, 4E-BP1/2 wild type (left) or 4E-BP1 knockout (right), using 4E-BP1 (53H11) Rabbit mAb. 4E-BP wild type and knockout tissues kindly provided by Dr. Nahum Sonenberg, McGill University.
Learn more about how we get our imagesConfocal immunofluorescent analysis of HeLa cells using 4E-BP1 (53H11) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Learn more about how we get our imagesConfocal immunofluorescent analysis of 293 cells, expressing either nontargeting shRNA (left) or shRNA targeting 4E-BP1/2 (right), using 4E-BP1 (53H11) Rabbit mAb #9644 (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4084.
Learn more about how we get our imagesFlow cytometric analysis of COS cells using 4E-BP1 (53H11) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
Learn more about how we get our imagesFor 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 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 October 2017
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 Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted February 2010
revised March 2016
Protocol Id: 283
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2017
Protocol Id: 404
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
Immunohistochemistry (Paraffin) | 1:2400 |
Immunofluorescence (Immunocytochemistry) | 1:1600 |
Flow Cytometry | 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.
4E-BP1 (53H11) Rabbit mAb detects endogenous levels of total 4E-BP1 protein.
Human, Mouse, Rat, Monkey
4E-BP1 (53H11) Rabbit mAb is produced by immunizing rabbits with a synthetic peptide corresponding to residues surrounding Ser112 of human 4E-BP1.
Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Alexa Fluor is a registered trademark of Life Technologies Corporation. DRAQ5 is a registered trademark of Biostatus Limited. U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
Explore pathways related to this product.
Product # | Size | Price |
---|---|---|
9644T | 20 µl (2 western blots) | $ 109.0 |
9644S | 100 µl (10 western blots) | $ 255.0 |