Protein extracts were made by trichloroacetic acid (TCA) extraction and analyzed by western blotting as described previously (Pai et al., 2014 (link)). TAP-tagged proteins were detected with peroxidase-antiperoxidase-soluble complex (P1291; Sigma). Cdc22-GFP was detected using antibody 1181446000 (Roche), and α-tubulin was detected with antibody T5168 (Sigma). Phos-tag Acrylamide gel was used to detect Cds1-P (Wako).
Phos tag acrylamide gel
Manufactured by Fujifilm
Sourced in United States
Phos-tag acrylamide gels are a specialized electrophoresis product designed for the detection and analysis of phosphorylated proteins. The gels incorporate a unique Phos-tag ligand that selectively binds to phosphorylated amino acid residues, allowing for the separation and visualization of phosphorylated protein species.
Automatically generated - may contain errors
Lab products found in correlation
P1291, by Merck Group (1 mentions)
T5168, by Merck Group (1 mentions)
λ ppase, by New England Biolabs (1 mentions)
Po448, by Agilent Technologies (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Iblot apparatus, by Thermo Fisher Scientific (1 mentions)
Anti rabbit secondary antibody conjugated to horseradish peroxidase, by Merck Group (1 mentions)
9 protocols using phos tag acrylamide gel
1
TCA Extraction and Western Blotting for Protein Analysis
2
In Vivo Phosphorylation Analysis
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For in vivo phosphorylation, protoplast isolation and DNA transfection were performed as described previously (67 (link)). After transfection with the plasmids and a subsequent 12-h incubation at room temperature to enable protein expression, cells were collected, and proteins were extracted using the IP buffer as described above for the Co‐IP assay. Total protein samples were separated on a Phos-tag acrylamide gel (25 μM, Wako Chemicals). Immunoblotting analysis was performed using HRP-conjugated anti-Flag or HRP-conjugated anti-HA antibodies.
For the dephosphorylation of RAPTOR1B-CT, 3 × Flag-tagged RAPTOR1B-CT protein was isolated using an extraction buffer containing 50 mM HEPES (NaOH, pH 7.5), 150 mM NaCl, 2 mM DTT, 0.5% Triton X-100, and 10% glycerol, supplemented with EDTA-free protease inhibitor (Roche). After centrifugation at 15,000 g for 10 min, the supernatant was incubated with 10 μL prewashed anti-Flag M2 agarose beads for 1 h at 4 °C on a roller shaker. The beads were washed twice with the extraction buffer and once with 1 × dephosphorylation buffer provided in a lambda protein phosphatase package (λ-PPase, New England Biolabs). The protein-bound beads were then directly used for phosphatase reactions following the manufacturer’s instructions. Proteins were eluted using 1 × SDS–PAGE loading buffer and boiled for 5 min before being separated on a Phos-tag gel.
For the dephosphorylation of RAPTOR1B-CT, 3 × Flag-tagged RAPTOR1B-CT protein was isolated using an extraction buffer containing 50 mM HEPES (NaOH, pH 7.5), 150 mM NaCl, 2 mM DTT, 0.5% Triton X-100, and 10% glycerol, supplemented with EDTA-free protease inhibitor (Roche). After centrifugation at 15,000 g for 10 min, the supernatant was incubated with 10 μL prewashed anti-Flag M2 agarose beads for 1 h at 4 °C on a roller shaker. The beads were washed twice with the extraction buffer and once with 1 × dephosphorylation buffer provided in a lambda protein phosphatase package (λ-PPase, New England Biolabs). The protein-bound beads were then directly used for phosphatase reactions following the manufacturer’s instructions. Proteins were eluted using 1 × SDS–PAGE loading buffer and boiled for 5 min before being separated on a Phos-tag gel.
3
In Vitro Phosphorylation Analysis of YDA
4
Chk1 and Cds1 Phosphorylation Analysis
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Proteins were extracted by TCA method and resolved on 3–8% Tris Acetate NuPAGE (Thermo Scientific) in reducing conditions (NuPAGE Tris Acetate SDS running buffer–Thermo Scientific) [80 (link)]. For analysis of Chk1 and Cds1 phosphorylation, cells were grown to log phase in YES at 30°C. As a positive control for Chk1 phosphorylation, cells were grown in YES plus 0.025% MMS. As a positive control for Cds1 phosphorylation, cells were grown in YES plus 10 mM hydroxyurea. In the case of P-Chk1 analysis, proteins were resolved by SDS-PAGE using 10% gels with an acrylamide/bisacrylamide ratio of 99:1 [81 (link)]. Phos-tag acrylamide gels (Wako) were used to detect Cds1-P. Proteins were transferred onto a PVDF membrane by a dry transfer method using the iBlot2 Dry Blotting System (Thermo Fisher Scientific). Every step from blocking to washes to antibody incubation was performed via sequential lateral flow with the iBind Flex Western device. HA-tagged proteins were detected with the rabbit monoclonal antibody HA-Tag (C29F4). The secondary anti-rabbit used was Dako PO448. α tubulin was used as a loading control and the probing was done by using mouse monoclonal anti-α-tubulin (Sigma T5168). The secondary antibody used was goat anti-mouse (HRP) Ab97040.
5
Detecting Phosphorylated Proteins Using Phos-tag SDS-PAGE
6
Detecting Phosphorylated Proteins Using Phos-tag SDS-PAGE
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To detect phosphorylated proteins, SDS-PAGE with Phos-tag acrylamide gels (Wako chemicals) were used. After electrophoresis, Phos-tag acrylamide gels were washed using transfer buffer with 5 mM EDTA for 10 min with gentle shaking and then replaced with transfer buffer without EDTA for 10 min according to the manufacturer’s protocol. Proteins were transferred to PVDF membranes and detected by the indicated antibodies using standard immunoblotting procedures.40 (link)
7
Phosphorylation Analysis of Smc4 and Ycg1
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Eight percent Phos-tag acrylamide gels (Wako Chemicals USA) were used to resolve Smc4-NT phosphorylated species (Kinoshita et al. 2006 (link)), whereas gels containing 7.5% Next gel acrylamide (Amresco) were used to monitor Smc4 abundance and Ycg1 phosphorylation (St-Pierre et al. 2009 (link)). All gels were transferred using the iBlot apparatus (Invitrogen). Antibodies and conditions used for immunoblotting are listed in the Supplemental Material.
8
Phos-tag Acrylamide Gel Electrophoresis for Phosphoprotein Analysis
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Phos-tag acrylamide gels were prepared according to the instructions provided (Wako); 10% acrylamide gels were copolymerized with 25 nM Phos-tag acrylamide and 10 nM MnCl2. 20-ml of bacterial cultures were centrifuged at 5,000 g at 4 °C for 10 min, and the pellets suspended in 1 ml 10 mM Tris pH 8.0. The cells were lysed using a French pressure cell (18,000 lb/in2). Samples were stored on ice prior to loading onto Phos-tag acrylamide gels and run at 4 °C. Gels were fixed for 10 min in transfer buffer with 10 mM EDTA and then washed for 10 min in transfer buffer without EDTA twice. After transfer to a nitrocellulose membrane, the samples were probed with rabbit polyclonal anti-Spo0A or anti-FliC antibodies at a 1:1000 dilution and an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Sigma) was used at dilution 1:10,000. Images were adjusted and cropped and quantified using ImageJ (http://rsbweb.nih.gov/ij/ ). In order to dephosphorylate Spo0A ~ P, samples were incubated at 100 °C for 5 min.
9
Phosphorylated HipT Separation Assay
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15% Phos-tag acrylamide gels (Wako) were cast according to the manufacturer’s guidelines, except that 100 µM Phos-tag acrylamide was added to ensure proper separation between phosphorylated and unphosphorylated HipT. The unphosphorylated inactive HipTS57A+D210A control was expressed from pSNN2. The gel was run at 4°C until the loading dye reached the bottom of the gel and visualized using standard Coomassie Blue staining as for normal SDS–PAGE gels.
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