We used the PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit (Catalog No. 8803, Cell Signaling Technology, United States) for immunoaffinity purification of tyrosine phosphopeptides. Immunoaffinity purification of tyrosine phosphopeptides was carried out as per the manufacturer’s protocol. Briefly, lyophilized peptides were dissolved in IAP buffer containing 50 mM MOPS, pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl. Before phosphotyrosine enrichment, the P-Tyr-1000 beads were washed twice with IAP buffer at 4°C. The peptide mixture was then incubated with P-Tyr-1000 beads for 30 min with gentle rotation. To remove nonspecifically bound peptides, the beads were washed thrice with ice-cold IAP buffer and twice with ice-cold water. The elution of enriched peptides from the beads was carried out at room temperature using 0.15% TFA. This step was repeated twice. This was followed by the cleanup of the samples using C18 StageTips.
Ptmscan phospho tyrosine rabbit mab p tyr 1000 kit
Manufactured by Cell Signaling Technology
Sourced in United States
The PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit is a laboratory tool used for the identification and analysis of phosphorylated tyrosine residues in proteins. It contains a highly specific antibody that recognizes over 1,000 tyrosine-phosphorylated peptides, enabling comprehensive detection of tyrosine phosphorylation events.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ary003b, by R&D Systems (1 mentions)
Imac select affinity gel, by Merck Group (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Antibiotic antimitotic solution, by Thermo Fisher Scientific (1 mentions)
Camkk2, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle medium dmem with high glucose, by Thermo Fisher Scientific (1 mentions)
Tpck treated trypsin, by Worthington (1 mentions)
Rslcnano, by Thermo Fisher Scientific (1 mentions)
Tmt10plex isobaric label reagent set, by Thermo Fisher Scientific (1 mentions)
Ptmscan kit, by Cell Signaling Technology (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
7 protocols using ptmscan phospho tyrosine rabbit mab p tyr 1000 kit
1
Tyrosine Phosphopeptide Enrichment Protocol
2
Quantitative Phosphoproteomics of SW620 Cells
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Quantitative phosphoproteomics was performed as in [31 ]. Briefly, SW620 cells were lysed in urea buffer (8 M urea in 200 mM ammonium bicarbonate pH 7.5). Phosphopeptides were purified after tryptic digestion of 20 mg (for cells) or of 35 mg (for mouse tumors) total proteins using the PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit (Cell Signaling Technology), according to manufacturer’s protocol. An additional enrichment step using the IMAC-Select Affinity Gel (Sigma Aldrich) was performed to increase the phosphopeptide enrichment. Data are available via ProteomeXchange with identifier PXD030006. Purified phosphopeptides were resuspended in 10% formic acid and two technical replicates for each sample were analyzed. Phospho-kinase arrays: proteome profiler human phospho-kinase array including phosphorylation of 43 kinases (ARY003B) and human phospho-RTK array (ARY001B) kits including 49 RTKs were purchased from R&D Systems. Indicated SW620 cells were lysed, and 300 μg of protein lysates were subjected to western blotting according to the manufacturer’s protocol. Signals on membranes were quantified using the Amersham Imager 600 (GE Healthcare) from 2 independent biological replicates.
3
Phosphotyrosine Peptide Enrichment for SILAC Proteomics
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Cell lysis was accomplished in urea buffer (8 m urea, 50 mm Hepes, pH 7.6) using 2 ml buffer for 1 × 108 cells. Ten milligrams of total cell lysate of each sample (light, medium and heavy labeled) were mixed to have a final amount of 30 mg SILAC sample for peptide preparation. Phosphotyrosine peptides were enriched using a mixture of two phosphotyrosine antibodies: The pY1000 antibody that is part of the PTMScan Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit (Cell Signaling Technologies, Danvers, MA, USA; catalog number 8803), in addition to 50 μl NHS-coupled 4G10 antibody. For the pY eluates, a second step of phosphopeptide enrichment was performed on TiO2 tips as described23 (link) prior to mass spectrometry (MS) analysis.
4
Phospho-Tyrosine Protein Kinase Assay
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The PTMScan® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit, AMPK, phospho-AMPK at T172, CAMKK2, PTK2, phospho-PTK2 at Y925, phospho STAT3 at Y705, C-Jun, phospho C-Jun at T73 antibodies were obtained from Cell Signaling Technology (Danvers, United States). Anti-β actin–HRP conjugated antibody was purchased from Sigma-Aldrich, United States; STO-609 (7-oxo-7H-benzimidazo [2,1-a]benz[de] isoquinoline-3-carboxylic acid–acetic acid), a CAMKK2 inhibitor, was purchased from Santa Cruz Biotechnology, Inc. (Texas, United States). TPCK-treated trypsin was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, fetal bovine serum (FBS), and antibiotic–antimitotic solution were purchased from Gibco (Thermo Fisher Scientific, United States). All other consumables used in the study were from Sigma Aldrich, United States.
5
Quantitative Phosphoproteomics of RXDX-106 Treatment
6
Phosphotyrosine Peptide Enrichment and Identification
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Immunoaffinity enrichment of phosphotyrosine peptides was accomplished with PTMScan ® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit (Cell Signaling), as indicated in the Supplemental Methods. Samples containing phospho-Tyr peptides were loaded in a nano Easy-nLC 1000 chromatographic system (Proxeon), and mass spectra acquired on an LTQ-Orbitrap Velos (ThermoScientific). The spectra were searched against the human SwissProt database and validation of phosphorylation sites was performed by ptmRS and Percolator algorithms.
Protein p-sites included in the analysis were those identified by at least two peptide spectrum matches (PSMs). Fold changes were calculated as the ratio of the number of PSMs that led to the identification of the corresponding protein p-site for the two compared conditions. Further details on the mass spec analysis and phosphosite identification can be found in the Supplemental Methods section.
Protein p-sites included in the analysis were those identified by at least two peptide spectrum matches (PSMs). Fold changes were calculated as the ratio of the number of PSMs that led to the identification of the corresponding protein p-site for the two compared conditions. Further details on the mass spec analysis and phosphosite identification can be found in the Supplemental Methods section.
7
Enrichment of TiO2 and Phosphotyrosine Peptides
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For TiO2 enrichment, titanium dioxide beads (Canadian Life Science) were resuspended in binding solution (80% Acetonitrile, 3% trifluoroacetic acid (TFA), 290 mg/mL DHB (Sigma) to have a final concentration of 200 μg/μL. Digested peptides are also resuspended in 200 μL of binding solution. Bead slurry (beads + binding solution) is added to the peptide solution in 1:2 ratio of protein:beads (3 mg of peptides:6 mg of beads). Peptide–bead solution is incubated on a rotator for 30 min at RT. Beads were then centrifuged at 5000 × g for 1 min and washed 3× with 75 μL of 30% acetonitrile (ACN) and 3% TFA. Another 3 washes were carried out with 75 μL of 80% ACN and 0.3%TFA. Beads are then eluted 2× with 75 μL of 15% NH4OH and 40% ACN. Eluted peptides are then dried by speed vacuuming for 2 h. For phosphotyrosine enrichment, PTMScan® Phospho-Tyrosine Rabbit mAB (p-Tyr-1000) Kit (Cell Signaling) was used according to the manufacturer’s instructions. TiO2 enrichment and pY100 immunoaffinity precipitation were done in two biological independent replicates.
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