For SDS–PAGE, protein samples were subjected to the gel electrophoresis at room temperature (12% acrylamide, 375 mM Tris–HCl, pH 8.8, and 0.1% SDS) followed by transfer blotting onto nitrocellulose membrane. For Phos‐tag SDS–PAGE, proteins from kinase assays were subjected to Phos‐tag gel electrophoresis [6% acrylamide, 375 mM Tris–HCl, pH 8.8, 50 μM Phos‐tag (Wako), and 100 μM MnCl2] at 4°C followed by transfer blotting. After incubation with the primary and secondary antibodies, the immuno‐blots were exposed and observed using a Bio‐Rad Image Analyzer. Relative protein levels were quantified with the Image Lab software (Bio‐Rad).
Phos tag
Manufactured by Fujifilm
Sourced in Japan, Germany, United States, China
Phos-tag is a chemical reagent used in affinity purification and detection of phosphorylated proteins. It binds selectively to phosphorylated amino acid residues, enabling identification and isolation of phosphorylated proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Nitrocellulose, by Bio-Rad (4 mentions)
Phosstop, by Roche (3 mentions)
Anti flag antibody, by Merck Group (3 mentions)
Total erk2, by Santa Cruz Biotechnology (2 mentions)
Slc12a2, by Cell Signaling Technology (2 mentions)
Slc12a4, by Abcam (2 mentions)
Can get signal solution, by Toyobo (2 mentions)
Lambda protein phosphatase, by Fujifilm (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Anti ha, by Merck Group (2 mentions)
λ protein phosphatase, by New England Biolabs (2 mentions)
Anti flag, by Merck Group (2 mentions)
Image studio software, by LI COR (2 mentions)
Lambda protein phosphatase λpp, by New England Biolabs (2 mentions)
λ phosphatase, by New England Biolabs (2 mentions)
Amersham typhoon 5 biomolecular imager, by GE Healthcare (2 mentions)
γ 32p atp, by Hartmann Analytic (2 mentions)
138 protocols using phos tag
1
Protein Separation and Quantification
2
Tissue Lysis and Phosphorylation Analysis
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Several different tissues from an 8 week old male mouse that had been fed with normal chow under regular day-light and dark cycle were directly lysed in buffer-L (20 mM HEPES-KOH, pH 7.4, 0.15 M NaCl, 25 μg/ml each of leupeptin and antipain, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol) containing 0.5% Nonidet P-40% and 0.1% SDS by ten strokes of homogenization with an Elvehjem-Potter homogenizer (Miura et al., 1992 (link)). After centrifugation, solubilized fractions (15 µg) were subjected to SDS-PAGE as described (Natsuyama et al., 2013 (link)). In phosphatase treatment, the soluble fractions were incubated with 1 µg/ml of λ-protein phosphatase (New England Biolab) for 30 min at 30 °C. Phos-tag PAGE was performed with 7.5% polyacrylamide gels containing 50 µM Phos-tag (Wako Chemicals) and 100 µM MnCl2 for the lysates of mouse tissues and 25 µM Phos-tag and 50 µM MnCl2 for those of cultured cells (Kinoshita et al., 2006 (link)).
3
Phosphorylation Analysis of C. elegans Proteins
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Embryos were harvested from synchronized young adult worms and sonicated in 2% SDS, 65 mM Tris pH 7, 10% glycerol with protease and phosphatase inhibitors. Lysates were spun at 14,000 rpm for 30 min and cleared supernatants treated with 100 U alkaline phosphatase (Roche, Indianapolis, IN). Samples were run in parallel on Phos-tag gels (7% SDS-PAGE with 25 μM Phos-tag and 50 μM MnCl2, Phos-tag from Wako Chemicals, Japan) and 7% SDS-PAGE at 30 mA for 2.5 hr. Gels were washed in transfer buffer with 1 mM EDTA twice for 10 min each and washed in transfer buffer without EDTA twice for 10 min each. Western blot transfer was performed for 1 hr at 4°C onto nitrocellulose membranes. Membranes were blocked and washed in 5% milk, 0.1% Tween-20 in PBS and probed with JL-8 antibody (1:240 dilutions, Clontech).
4
Phosphoprotein Analysis of Mouse Tissues
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Several different tissues from an 8-week old male mouse that had been fed with normal chow under regular day-light and dark cycle were directly lysed in buffer-L (20 mM HEPES-KOH, pH 7.4, 0.15 M NaCl, 25 μg/ml each of leupeptin and antipain, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol) containing 0.5% Nonidet P-40 and 0.1% SDS by ten strokes of homogenization with an Elvehjem-Potter homogenizer (Miura et al., 1992) . After centrifugation, solubilized fractions (15 µg) were subjected to SDS-PAGE as described (Natsuyama et al., 2013) . In phosphatase treatment, the soluble fractions were incubated with 1 µg/ml of -protein phosphatase (New England Biolab) for 30 min at 30˚C. Phos-tag PAGE was performed with 7.5% polyacrylamide gels containing 50 µM Phos-tag (Wako Chemicals) and 100 µM MnCl 2 for the lysates of mouse tissues and 25 µM Phos-tag and 50 µM MnCl 2 for those of cultured cells (Kinoshita et al., 2006) .
5
Phos-tag Gel Immunoblotting Technique
6
Western Blot and Phos-tag Analysis
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Whole lysates were separated by SDS–PAGE and transferred to the PVDF membranes (Millipore). The blots were probed with indicated antibodies, and protein bands on the blot were visualized by the enhanced chemiluminescence reagent (Millipore). Phos‐tag‐PAGE was performed using 7% SDS–PAGE minigels containing 10 μM Phos‐tag (Wako) in the presence of 100 μM MnCl2 following the manufacturer's protocol.
7
Phosphoprotein Detection by Phos-tag SDS-PAGE
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Protein from integuments was extracted in ice-cold lysis buffer [150 mM NaCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 50 mM (pH 7.4) tris-HCl] with protease and phosphatase inhibitor mixtures (Roche). Protein samples were fractionated using 12% SDS-PAGE and transferred onto a nitrocellulose membrane (Merck Millipore). In addition, Phos-tag SDS-PAGE gel was prepared by adding an additional 100 μM MnCl2 and 50 μM Phos-tag (Wako, Japan) to the 12% SDS-PAGE. Phos-tag SDS-PAGE gels were washed twice by gently shaking in transfer buffer containing 10 mM EDTA for 10 min and incubated in transfer buffer without EDTA for 10 min before being transferred to polyvinylidene fluoride membranes (Merck Millipore). After blocking with 3% BSA, the membrane was incubated with the corresponding primary antibodies and subsequently with HRP-conjugated secondary antibodies (Proteintech) and visualized using the chemiluminescent nucleic acid detection module (Thermo Fisher Scientific). The blotting band intensity was determined using ImageJ software (NIH, Bethesda, MD, USA). It should be noted that Phos-tag SDS-PAGE can detect phosphorylated and nonphosphorylated proteins by their band shift differences.
8
Phosphate-Binding Gel Electrophoresis Protocol
9
Western Blotting and Phos-tag Gel Electrophoresis
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Protein samples for Western blotting were boiled at 95 °C for 5 min in 2× SDS sample buffer and separated on standard Lämmli SDS-PAGE gels. Proteins were semidry blotted onto polyvinylidene fluoride (PVDF) membranes (Roche), which were subsequently blocked in 5% milk in PBST, incubated with primary antibodies in 5% milk in PBST over night at 4 °C, followed by three PBST washes, incubation with the secondary antibodies for 1 h in 5% milk in PBST and final three washes with PBST. Chemiluminescence signals were detected using the imager ImageQuant LAS4000mini (GE Healthcare) or IQ800 (Cytiva) or a film developer machine.
Phos-tag (WAKO) gel electrophoresis for analysis of phosphorylated SAP102 as well as the sample preparation for phosphatase treatment was done as described before (55 ) using Bis-Tris–buffered neutral pH gels with 6% polyacrylamide supplemented with 20 to 75 μM Phos-tag.
Phos-tag (WAKO) gel electrophoresis for analysis of phosphorylated SAP102 as well as the sample preparation for phosphatase treatment was done as described before (55 ) using Bis-Tris–buffered neutral pH gels with 6% polyacrylamide supplemented with 20 to 75 μM Phos-tag.
10
Phosphorylation Analysis of Myosin-A in Intracellular Parasites
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Intracellular parasites 24 hours post-infection were harvested in intracellular buffer [31 (link)], filtered with 3-μm Nucleopore membrane, pelleted and re-suspended in either intracellular buffer or extracellular buffer [31 (link)]. Parasites were then incubated at 37°C for 2 minutes and immediately placed on ice followed by centrifugation at 1000 g for 10 min at 4°C. The parasite pellet was then lysed with RIPA buffer containing phosphatase inhibitor, PhosSTOP (Roche) followed by addition of SDS sample buffer containing β-meracaptoethanol and heated at 100°C for 5 min. To examine phosphorylation status of Myosin-A, Phos-tag gel electrophoresis was carried out according to manufacturers instructions (Wako Chemicals, USA). Briefly 200 μM Phos-tag (Wako Chemicals, USA) and 100 μM MnCl2 were added to conventional 7.5% (w/v) acrylamide resolving gel and the gel was run at constant voltage at RT. The gel was washed three times in SDS-PAGE running buffer containing 10 mM EDTA and once each in running buffer and transfer buffer before transferring to a PVDF membrane for immunoblotting using anti-MyosinA antibody.
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