Tyrosine phosphorylation, a reversible process, is one of the most frequent post-translational modifications of proteins and is crucial in mediating signal transduction in eukaryotic cells after exposure to cytokines and growth factors (1). Anti-phosphotyrosine antibodies have been an important tools in studying the level of tyrosine phosphorylation of proteins in different cellular models. It has also played an important role in enriching phosphotyrosine peptides from trypsin-digested cell lysates. As a result, a large number of phosphopeptides have been identified under various physiological and pathological conditions with mass spectrometry technologies (2-3).
The following protocols provide optimized techniques for detecting proteins with a phosphotyrosine residue in Western blot, Immunoprecipitation, Immunfluorsecence, and ELISA techniques.
Use as indicated in method at 1:500 dilution, sufficient for 100 ml of working strength Ab.
1. Run protein samples and control samples on SDS-PAGE.
2. Equilibrate the gel in Western blot buffer (25 mM Tris pH 8.3, 192 mM glycine, 5% methanol) for 15 min at room temperature prior to electro-blotting.
3. Transfer the protein to a PVDF membrane overnight at 40V at 4oC.
4. Wash the membrane once with TBST for 10 minutes (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20).
5. Block the membrane surface with 5% nonfat-dry milk in TBST for 60 min at room temperature with constant agitation.
6. Incubate the membrane with a 1:500 dilution of anti-phosphotyrosine (Cat. # APY03) antibody diluted in TBST for 1h at room temperature or overnight at 4°C with constant agitation.
7. Wash the membrane 3 times in TBST for 10 min each.
8. Incubate the membrane with an appropriate dilution (e.g., 1:20,000) of anti-mouse secondary antibody (e.g., goat anti-mouse HRP conjugated IgG from Jackson Labs., Cat. # 115-035-068) in TBST/3% non-fat milk for 30 min at room temperature.
9. Wash the membrane 6 times in TBST for 10 min each.
10. Use an enhanced chemiluminescence detection method to detect the signal (e.g., SuperSignal West Dura Extended Duration Substrate; ThermoFisher).
Western Blot Demonstration of APY03 Phosphotyrosine Specificity
Legend: A431 cells were either treated (+) or untreated (-) with Calyculin A (CalyA: a serine/threonine phosphatase inhibitor, 50nM for 1 hour). NIH3T3 cells were either treated or untreated with H2O2-activated sodium orthovanadate (Vanadate: a specific tyrosine phosphatase inhibitor, 100 µM for 10 minutes). 16 µg of each lysate was resolved in SDS-PAGE and proteins were transferred to PVDF membrane. Membranes were probed as follows:
APY03 primary Ab: Detected tyrosine phosphorylated proteins in Vanadate-treated cells but not serine or threonine phosphorylated proteins in CalyA-treated cells.
Anti-phosphoserine & anti-phosphothreonine primary Ab: Detected serine/threonine phosphorylated proteins in CalyA-treated cells, demonstrating that phosphoserine/threonine proteins are present in CalyA treated lysates but are not detected by APY03. The anti-phosphoserine and anti-phosphothreonine Abs are from Millipore, Cat# AB1603 & AB1607 respectively.
Use as indicated at 5 µl per IP reaction (200-500 µg total lysate per IP), sufficient for approximately 40 IP assays.
1. Add 5 µl of anti-phosphotyrosine (Cat. # APY03) to 500 µl of PBS pH 7.4 in a microfuge tube containing 30 µl of packed protein G agarose pre-equilibrated in PBS.
2. Gently rotate the reaction for 1 hour at 4°C.
3. Add 500 µl of PBST to the mixture and centrifuge for 1 minute at 4°C and 3000 rpm (960 x g). Addition of the PBST will prevent agarose from sticking to the microfuge tube walls.
4. Discard supernatant and wash beads 3X in PBST (500 µl per wash).
5. Add 200-500 µg of cell lysate (0.5 mg/ml protein in RIPA buffer) to the beads.
6. Gently rotate the reaction at 4°C for 1 hour.
7. Spin down agarose for 1 minute in 4°C at 3000 rpm (960 x g).
8. Discard supernatant and wash beads with 800ul of wash buffer (50mM Tris pH7.5, 150mM NaCl and 1% IGEPAL). Spin down agarose for 1 minute in 4°C at 3000 rpm (960 x g).
9. Repeat wash two more times. Carefully remove all supernatant without disturbing beads.
10. Resuspend beads in 30 µl of 2X non reducing SDS sample buffer and incubate at room temperature for 5 minutes.
11. Spin down bead for 1 minute at 14,000 rpm (21,000 x g). Carefully remove 25 µl of supernatant and transfer to a new tube and add 1 µl of 2-mercaptoethanol. Boil for 5 minutes.
12. Load samples on SDS-PAGE for subsequent western blot analysis. NOTE: to avoid detection of Ab heavy and light chains, we recommend using HRP conjugated CleanBlot (Thermo Scientific, #21230) as the secondary antibody reagent.
IP of pervanadate-treated lysates from NIH3T3 cells
Legend: NIH3T3 cells were either treated (+) or untreated (-) with H2O2/orthovanadate (100 µM for 10min). Cell lysate was prepared in RIPA buffer and 200 µg of lysate per reaction was used for immunoprecipitation of tyrosine-phosphorylated proteins. APY03 was first bound to protein G beads and then incubated with cell lysate. For bead only control, cell lysate was incubated with protein G beads without APY03. Western blots of immunoprecipitated proteins were developed using APY03 at 1:500 dilution and CleanBlot (Thermo Scientific, #21230) as secondary antibody. No IP input lysate represents the signal from 5% of H2O2/orthovanadate treated or untreated NIH3T3 lysate. As shown in Figure 2, APY03 was able to enrich a wide range of tyrosine-phosphorylated proteins from NIH3T3 cells treated with H2O2-activated orthovanadate. No signal was detected with Protein G bead control without APY03.
Use as indicated below at 1:500-1:1000 dilution, sufficient for 100-200 ml of working strength Ab, approx. 1000-2000 IF slides.
1. Plate cells to required density in suitable growth media on acid washed coverslips in tissue culture dish.
2. Allow cells to grow for 24-48 hours then treat one set of coverslips with EGF (100ng/ml for 3 minutes, A431 cells), the other with H2O2-activated sodium orthovanadate (100 µM for 10 minutes, NIH3T3 cells).
3. Fix cells by dipping coverslips in -20°C methanol for 5 minutes (it is convenient to use a porcelain coverslip rack for fixing and rinsing samples).
4. Remove coverslips and dip into -20°C acetone for 2 minutes.
5. Remove coverslips and air dry coverslips at room temperature.
6. Rehydrate cells by dipping coverslips in PBS and then place coverslips, cell side up, on parafilm and apply 100-200 µl APY03 solution (1:500 in PBS for A431 cells and 1:1000 in PBS for NIH3T3 cells).
7. Incubate at room temperature for 60 minutes.
8. Wash coverslips three times in PBS at room temperature over a 5 minute period.
9. Apply fluorescently labeled anti-mouse secondary antibody at manufacturers recommended dilution. For example, we use Alexa Fluor 555 goat anti-mouse (Life Technologies, Cat# A-21424) at 1:500 dilution in PBS.
10. Incubate at room temperature for 60 minutes.
11. Wash coverslips three times in PBS at room temperature over a 5 minute period.
12. Rinse coverslips briefly in sterile water.
13. Dip coverslips in absolute ethanol at room temperature for 2 minutes and transfer to blotting paper, cell side up, and allow to dry.
14. Place coverslips, cell side down, on glass slide with mounting media (e.g. EMS, Cat# 17987-10) and observe cells under fluorescence microscope.
IF detection of phosphotyrosinated proteins in vanadate-treated cells
Legend: Human epidermoid carcinoma A431 cells, untreated (3A) or treated (3B) with EGF (100 ng/ml for 3 minutes), and NIH3T3, untreated (3C) or treated (3D) with H2O2-activated sodium orthovanadate (100 µM for 10 minutes), were stained as described in the method. Phosphotyrosine and nuclei were visualized in green fluorescence and blue DAPI staining, respectively.
Use as indicated below at 1:10,000 dilution, sufficient for approximately 40,000 assays.
1. Add 40 ng in 50 µl PBS of phosphopeptide BSA to the wells of ELISA plate.
2. Incubated plate overnight at 4oC.
3. Wash plate 2 times with 200 µl PBS.
4. Add 200 µl of 3%milk in PBS and incubate at room temperature for 1 hour.
5. Wash plate 3 times with 200 µl PBS.
6. Add 50 µl of diluted APY03 (1:10,000 dilution in PBS). Incubated at room temperature for 1 hour with shaking.
7. Wash plate with 3 times with 200 µl PBST.
8. Add 50 µl of diluted goat anti-mouse secondary antibody (1:3000 dilution in 3%milk/PBST). Incubate at room temperature for 1 hour with shaking.
9. Wash plate 3 times with 200 µl PBST.
10. Add 50 µl of substrate to each well. Stop reaction by adding 50 µl of stop solution.
11. Measure OD at 490nm.
ELISA assay demonstrating specificity of APY03 for phosphotyrosine
Legend: BSA was modified by conjugating with a library of phosphopeptides. 40ng of each phosphotyrosine, phosphothreonine and phosphoserine BSA were added to a individual well of a ELISA plate. APY03 at 1:10,000 dilution and goat anti mouse secondary antibody at 1:3000 were used to develop signal. Data shows that APY03 does not crossreact with phosphothreonine and phoophoserine modified BSA.
Sodium pervanadate should be made fresh and used immediately. Pervanadate is made by adding 6 µl of hydrogen peroxide to 44 µl of PBS in a microfuge tube. Add 50 µl of 200 mM sodium orthovanadate (the solution will turn yellow) and use immediately at the recommended dilutions.
SysPTM: A Systematic Resource for Post-Translational Modification
What you can do
SysPTM provides a systematic and sophisticated platform for proteomic PTM research, equipped not only with a knowledge base of manually curated multi-type modification data, but also with four fully developed, in-depth data mining tools.
RegPhos: Regulatory Network in Protein Phosphorylation
What you can do
RegPhos is a resource to explore the protein kinase-substrate phosphorylation networks in human and mouse.
1. Machida, K. et al. (2003) Profiling the global tyrosine phosphorylation state. Mol. Cell. Proteomics 2, 215-233
2. Blagoev, B. et al. (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 22, 1139-1145
3. Schmelzle, K. et al. (2006) Temporal dynamics of tyrosine phosphorylation in insulin signaling. Diabetes 55, 2171-2179