Phosphate-affinity SDS-PAGE was performed by using Phos-tag SDS-PAGE as described previously (59 (link)). Briefly, cell lysates were prepared using cell lysis buffer without inhibitors (Beyotime). The proteins were separated using Phos-tag SDS-PAGE that was performed with 7% polyacrylamide gels containing 50 μM Phos-tag AAL-107 (Wako) and 100 μM MnCl2 under constant-current conditions (25 mA/gel). The gels were equilibrated with transfer buffer supplemented with 10 mM EDTA via three 10-min washes with shaking and then equilibrated with transfer buffer lacking EDTA via an additional 10-min wash before being subjected to a standard Western blotting procedure.
Phos tag aal 107
Manufactured by Fujifilm
Sourced in Japan
Phos-tag AAL-107 is a phosphate-affinity reagent that can be used to detect and isolate phosphorylated proteins. It functions by selectively binding to phosphorylated amino acid residues, allowing for the separation and analysis of phosphorylated proteins from complex biological samples.
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Lab products found in correlation
Lumiglo, by Cell Signaling Technology (1 mentions)
His tag, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Mouse anti v5, by Thermo Fisher Scientific (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Sc 7392 hrp, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Phosstop, by Roche (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Gel doc, by Bio-Rad (1 mentions)
Ez ecl, by Sartorius (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Lightcycler 480, by Roche (1 mentions)
9 protocols using phos tag aal 107
1
Phos-tag SDS-PAGE for Phospho-protein Analysis
2
Quantification and Detection of Tobacco Cell Proteins
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Proteins from tobacco cells were quantified using the Bradford method (Bradford, 1976 (link)) after disruption in lysis buffer, which consisted of 50 mM HEPES, pH 7.5, 50 mM EDTA, 2 mM dithiothreitol (DTT), 100 mM NaCl, and protease inhibitor cocktail (PIC) without EDTA (Roche), either supplemented or not with 10 mM N-Ethylmaleimide (NEM). Protein samples (20 μg) were resolved by 10–15% SDS-PAGE or 6% native-PAGE and visualized by immunoblotting with antibodies against CDC48 (Abcam), cAPX (Agrisera), or His-tag (Cell Signaling Technology). The immunoblots were examined using LumiGLO® (Cell Signaling Technology). After transfer, membranes were stained with Ponceau Red in order to check the loading of total proteins. For gel retardation assays, 12% gels were supplemented with 50 µM Phos-tag™ AAL-107 (Wako Pure Chemical Co.) and 100 µM MnCl2 and then rinsed in 1 mM EDTA for Mn2+ chelation.
3
Analyzing Phosphorylation of VGLUT1 Mutants
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All steps were performed at 4°C or on ice. Brains of wild type adult mice were dissected for the collection of brain regions. The samples were treated with homogenization buffer (0.32 M sucrose, 4 mM HEPES pH 7.4). Cultures expressing the different VGLUT1 mutants were collected on DIV 17 with 1 × PBS. Both buffers were supplemented with protease inhibitor cocktail (539134, Millipore) and Halt phosphatase Inhibitor Cocktail (78420, Thermo Fisher Scientific). When necessary, protein samples were treated with alkaline phosphatase prior to the biochemical analysis. FastAP Thermosensitive Alkaline Phosphatase (one unit/µl) and FastAP 10 × buffer (EF0654, Thermo Scientific) were added to samples and incubated for 1 hr at 37°C. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and phosphate affinity SDS-PAGE (Kinoshita et al., 2006 (link)) were conducted according to standard methods. For phosphate affinity SDS-PAGE (Mn2+-Phos-tag SDS-PAGE), 25 µM Phostag (AAL-107, Wako) and 0.1 mM MnCl2 were added to the resolving gel before polymerization. Western blotting was performed according to standard procedures using HRP-coupled secondary antibodies for qualitative detection. Chemi-luminescence signals were visualized with ChemiDoc MP System (Bio-Rad) using SuperSignal West Dura Extended Duration Substrate (34075, Thermo Scientific).
4
Phos-tag™ for Phosphorylated Protein Detection
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Phos-tag™ is a phosphate-binding reagent that is used for the separation, purification and detection of phosphorylated proteins (Kinoshita et al., 2006 (link)). We used this product (Phos-tag AAL-107, Wako Pure Chemical Industries) to detect phosphorylated proteins in SDS-PAGE gels. The samples were loaded into the wells of 8% SDS-PAGE with Mn2+-Phos-tag™ in a resolving gel. After electrophoresis, the gel was soaked in transfer buffer with 1 mM EDTA for 10 min and then in transfer buffer without EDTA for another 10 min before transfer onto a PVDF membrane using a wet-tank method followed by western blotting.
5
Phosphorylation detection of Pumilio protein
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S2 cells were lysed in CIP buffer (100 mM NaCl, 50 mM Tris- HCl pH 7.9, 10 mM MgCl, 1 mM DTT), 1 mM PMSF, 0.1% NP40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Calbiochem). For CIP treatment phosphatase inhibitor cocktail was omitted, and lysate was incubated at 37 °C, for 60 min in 2 units CIP (NEB) per 50 μL reaction containing 50μg of total protein. For λ-phosphatase (NEB) treatment, lysates were incubated with 1 unit of phosphatase for 30 min at 30 °C in λ−phosphatase buffer. Lysates were cleared by centrifugation and subjected to SDS-PAGE. To detect phosphorylated Pumilio in SDS-PAGE, we used Phos-tag AAL-107 (Wako Chemicals GmbH) according to the manufacturer’s instruction72 (link),73 (link). Western blotting was performed using mouse anti-V5 (1:5000, Invitrogen), followed by the corresponding Horseradish Peroxidase (HRP) conjugated secondary antibodies (1:5000, GE Healthcare) and visualized using the ECL Plus Western Blotting detection system (GE Healthcare).
6
Arabidopsis Protein Extraction and Detection
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For total protein extraction, Arabidopsis seedlings were grounded in the presence of liquid nitrogen to fine powder, and total protein was extracted with 2 × SDS sample buffer (100 mM Tris-Cl, pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, and freshly added 10% β-mercaptoethanol). Aliquots of denatured total protein were separated by SDS-PAGE and transferred to a PVDF membrane. For the detection of phosphorylated CBL proteins, total protein was separated by 10% SDS-PAGE with 15 μM Phos-tag (AAL-107, WAKO pure chemical industries, Ltd) and transferred to PVDF membrane. For immunoblot analyses, anti-CBL3 (19 (link), 66 (link)), anti-GAPDH (PHYTOAB, PHY0303A), anti-actin (PHYTOAB, PHY0001), anti-Flag (Sigma-Aldrich, A8592-2MG), anti-S6K1-p (phosphor T449) (Abcam, ab207399), anti-HA (Santa Cruz Biotechnology, sc-7392HRP) were used as primary antibodies. Each experiment was repeated at least three times, and one representative result is shown in the figures. Band density in immunoblots was quantified using Image J software.
7
Phosphorylation Analysis via Phos-tag SDS-PAGE
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For sample preparation, cells were lysed using a pH neutral buffer (20 mM TrisHCl pH 8.1, 0.5% (v/v) NP-40, 200 mM NaCl, cOmplete EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP (Roche)) before Laemmli buffer was added. Gels were cast according to manufacturer’s protocol (wako-chem) using Phos-tag™ AAL-107 (MW: 595, Wako Cat. No. 304-93525). Samples were run on the Zn2+-Phos-tag SDS PAGE gels for 4 h at 60 V and were blotted over night at 4 °C onto nitrocellulose membranes (Hybond ECL, GE healthcare). Staining and analysis were done as described for standard SDS-PAGE. For the blots shown in Fig. 3d, e, the uncropped versions of the blots are provided in Supplementary Figure 8 .
8
Phos-tag Assay for Protein Phosphorylation
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Purified proteins were separated using Phos-tag gel technology (Wako, Phos-tag AAL-107). Samples were either expressed alone in BL21 cells and purified, treated with λPP protein after purification at 30°C for 1 hr (New England BioLabs, P0753L) or co-expressed in BL21 cells with RFP-lambda phosphatase. Purified protein samples were incubated in the presence or absence of ATP in kinase assay protein buffer (50 mM Tris, pH 8.5, 50 mM K acetate, 2 mM Mg acetate, 1 mM EGTA, 10% glycerol) with 1 mM DTT and 1 mM PMSF for 30 or 60 min at room temperature. Phos-tag SDS-PAGE was performed with pre-cast 7.5% polyacrylamide gels containing 50 μM Phos-tag acrylamide with MnCl2 (Wako, 192–18001). Electrophoresis was completed at 180 v for 90 min and the gel was stained with Coomassie blue. The stained gel was imaged using a GelDoc (BioRad) and the band intensity was quantified using ImageJ to draw a box over both the highest band and the lowest band in each lane. The measure of percent of protein that was phosphorylated was generated by dividing the intensity value of the highest band by the total intensity from the sum of the highest and lowest bands.
9
RNA Extraction and qRT-PCR for Gene Expression Analysis
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RNA was extracted using TRI Reagent (MRC, Cincinnati, OH, USA), PerfectPure RNA Purification kit (5 PRIME, Qiagen) or RNeasy (Qiagen) for PHH. First-strand synthesis was performed using qScript cDNA synthesis kit (Quanta, Beverly, MA, USA). qRT-PCR was performed using the LightCycler480 (Roche, Basel, Switzerland), with PerfeCta® SYBR Green FastMix mix (Quanta). All qPCRs were normalized to 18S mRNA levels or RPS11 (PHH). The primer sequences are in the supplementary materials .
Lysates were prepared from cells using RIPA buffer [20 (link)] supplemented with Dithiothreitol (DTT) and protease and phosphatase inhibitors (Sigma-Aldrich), and subjected to SDS-PAGE. Antibodies are listed in the supplements. We used enhanced chemiluminescence (ECL) detection using EZ-ECL (Biological Industries). For quantification of Western blot bands we used ImageJ (version 1.51k) software. (http://imagej.nih.gov/ij , accessed on 12 November 2018). Phos-Tag™-AAL-107 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and was added at 40 µM concentration to 6% SDS-PAGE gel.
Lysates were prepared from cells using RIPA buffer [20 (link)] supplemented with Dithiothreitol (DTT) and protease and phosphatase inhibitors (Sigma-Aldrich), and subjected to SDS-PAGE. Antibodies are listed in the supplements. We used enhanced chemiluminescence (ECL) detection using EZ-ECL (Biological Industries). For quantification of Western blot bands we used ImageJ (version 1.51k) software. (
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