Western blot analysis of extracts from NIH/3T3 cells untreated and treated with sodium vanadate (1 mM for 30 minutes), using 75 ng/ml Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated) (A), then detected by Anti-biotin Antibody, HRP-linked #7075. Western blot analysis of the same extract using 750 ng/ml Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411, then detected by Anti-mouse IgG, HRP-linked Antibody #7076 (B).
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, untreated (left) or λ-phosphatase-treated (right), using Phospho-Tyrosine mAb (P-Tyr-100) (Biotinylated).
Western blot analysis of extracts from NIH/3T3 cells untreated and treated with sodium vanadate (1 mM for 30 minutes), using 75 ng/ml Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated) (A), then detected by Anti-biotin Antibody, HRP-linked #7075. Western blot analysis of the same extract using 750 ng/ml Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411, then detected by Anti-mouse IgG, HRP-linked Antibody #7076 (B).
This product has been approved for use in this application by CST. No data images are currently available.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, untreated (left) or λ-phosphatase-treated (right), using Phospho-Tyrosine mAb (P-Tyr-100) (Biotinylated).
Storage

Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.

Western Blotting Protocol

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 product webpage for recommended antibody dilution.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix. Nonfat Dry Milk: (#9999). Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well. Wash Buffer: (#9997) 1X TBST. Bovine Serum Albumin (BSA): (#9998). Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well. Biotinylated Protein Ladder Detection Pack: (#7727). Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329). Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended. Streptavidin-HRP: (#3999). Detection Reagent: SignalFire™ ECL Reagent (#6883).B. Protein BlottingA general protocol for sample preparation. Treat cells by adding fresh media containing regulator for desired time. Aspirate media from cultures; wash cells with 1X PBS; aspirate. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). Heat a 20 µl sample to 95–100°C for 5 min; cool on ice. Microcentrifuge for 5 min.

Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: Loading of prestained molecular weight markers (#59329, 10 l/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 l/lane) to determine molecular weights are recommended.

Electrotransfer to nitrocellulose membrane (#12369).C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Wash three times for 5 min each with 15 ml of TBST.II. Primary Antibody Incubation Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C. Wash three times for 5 min each with 15 ml of TBST. Incubate membrane with Streptavidin-HRP (#3999 at the appropriate dilution) in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature. Wash three times for 5 min each with 15 ml of TBST. Proceed with detection (Section D).

Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.

D. Detection of ProteinsDirections for Use: Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.

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.

3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Streptavidin (Sepharose® Bead Conjugate) (For biotinylated antibodies): (#3419) Gently vortex vial and use 10 µl per immunoprecipitation. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.B. Preparing Cell Lysates Aspirate media. Treat cells by adding fresh media containing regulator for desired time. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice. Sonicate on ice three times for 5 sec each. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.C. ImmunoprecipitationCell Lysate Pre-Clearing (Optional) Add 10 µl Streptavidin (Sepharose® Bead Conjugate) (#3419; for biotinylated antibodies), to 200 µl cell lysate at 1 mg/ml. Incubate with rotation at 4°C for 30–60 min. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube. Proceed to immunoprecipitation below.Immunoprecipitation Add biotinylated antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C. Gently mix Streptavidin (Sepharose® Bead Conjugate) (#3419) and add 10 µl of slurry. Incubate with rotation for 2 hr at 4°C. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes. Proceed to sample analysis by western immunoblotting or kinase activity (section D).D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) 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).

For Analysis by Kinase Assay Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate. Incubate for 30 min at 30°C. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec. Transfer supernatant containing phosphorylated substrate to another tube. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g. Load the sample (15–30 µl) on SDS-PAGE (4–20%).
RECOMMENDED
DETECTION REAGENTS SignalStain Boost IHC Detection Reagent (HRP, Mouse) #8125 SignalStain Boost IHC Detection Reagent (AP, Mouse) #31926
Specificity / Sensitivity Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated) is prepared by biotinylation of the antibody via primary amines. Biotinylated antibody is useful for detection or purification of tyrosine-phosphorylated proteins using avidin-biotin binding. This antibody does not cross-react with proteins phosphorylated at serine or threonine. (U.S. Patent No\'s.: 6,441,140; 6,982,318; 7,259,022; 7,344,714; U.S.S.N. 11,484,485; and all foreign equivalents.)Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with phospho-Tyr-containing peptides . Antibody is purified by protein A chromatography.

Background Tyrosine phosphorylation plays a key role in cellular signaling (1). Research studies have shown that in cancer, unregulated tyrosine kinase activity can drive malignancy and tumor formation by generating inappropriate proliferation and survival signals (2). Antibodies specific for phospho-tyrosine (3,4) have been invaluable reagents in these studies. The phospho-tyrosine monoclonal antibodies developed by Cell Signaling Technology are exceptionally sensitive tools for studying tyrosine phosphorylation and monitoring tyrosine kinase activity in high throughput drug discovery. Schlessinger, J. (2000) Cell 103, 211-25.
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For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, 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 [email protected] For information regarding commercial licensing terms please contact CST Pharma Services Department at [email protected]

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