Phos‐tag™ acrylamide (Wako Pure Chemical Industries, Ltd.) was used for detection of mobility shift of phosphorylated proteins by SDS‐PAGE.22 , 23 Briefly, Phos‐tag™ acrylamide (60 μmol/L) and ZnCl2 (120 μmol/L) were added to a standard gel solution, which was polymerized by addition of ammonium persulfate and TEMED. To neutralize the effect of divalent metal chelators in the lysates used for Phos‐tag™ SDS‐PAGE, these were supplemented with ZnCl2 at a concentration equal to concentrations of previously added chelating agents. All subsequent procedures including electrophoresis, transfer and immunodetection were carried out by following standard protocol for Western blotting as described above.
Phos tag acrylamide
Manufactured by Fujifilm
Sourced in Japan, United States, Germany
Phos-tag acrylamide is a specialized laboratory equipment used for the detection and analysis of phosphorylated proteins. It functions as a phosphate-binding compound that can selectively capture and separate phosphorylated proteins from non-phosphorylated proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Mncl2, by Merck Group (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Ettan ipgphor 3, by GE Healthcare (2 mentions)
Immobiline drystrip ph 4 7, by GE Healthcare (2 mentions)
Amersham imager 600rgb, by GE Healthcare (2 mentions)
λ ppase, by New England Biolabs (2 mentions)
Bx795, by Cayman Chemical (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
Ecl plus, by GE Healthcare (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Donkey anti rabbit hrp, by Jackson ImmunoResearch (2 mentions)
Anti 6xhis antibody pa1 983b, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti mbp monoclonal antibody, by New England Biolabs (2 mentions)
Clarity ecl substrate, by Bio-Rad (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Lambda phosphatase, by New England Biolabs (2 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Laemmli buffer, by Boston BioProducts (2 mentions)
113 protocols using phos tag acrylamide
1
Phos-Tag Acrylamide for Phosphoprotein Detection
2
Phospho-affinity Gel Electrophoresis Using Phos-tag
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phospho-affinity gel electrophoresis was performed using Phos-tag acrylamide 7.5% (w/v) running gels polymerized with 50 μM Phos-tag acrylamide (FUJIFILM Wako Pure Chemical Corporation) and 100 μM MnCl2. Gel running and transfer conditions were optimized according to the manufacturer's protocol.
3
Phosphorylation Analysis of Rab7 Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phos-tag acrylamide (Wako Chemicals) based gel analysis was carried out to detect phosphorylated species of Rab7. PTEN-depleted HeLa cells were transfected with SFB-Rab7 wild type or SFB-Rab7 2PD mutants. Twenty-four hours post transfection, cells were lysed and subjected to immunoprecipitation using streptavidin–sepharose beads. The protein samples eluted by boiling were run on 12% polyacrylamide gel containing 50 μM Phos-tag acrylamide (Wako Chemicals) and 50 μM MnCl2. After electrophoresis, Phos-tag acrylamide gels were washed with gentle shaking in transfer buffer containing 1 mM EDTA for 15 min and then incubated in transfer buffer containing 0.01% SDS without EDTA for 15 min according to the manufacturer's protocol. Proteins were then transferred to polyvinylidene difluoride membrane for further analysis by standard immunoblotting protocol using specific antibodies.
4
Phospho-affinity Gel Electrophoresis for Protein Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phospho-affinity gel electrophoresis for mobility shift detection of phosphorylated proteins was performed using Phos-tag acrylamide 4.5% (w/v) running gels polymerized with 25 μM Phos-tag acrylamide (FUJIFILM Wako Pure Chemical Corporation) and 50 μM MnCl2. Gel running and transfer conditions were optimized according to the manufacturer’s protocol.
5
Phosphorylation of Vimentin by T. gondii
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phos-tag acrylamide (Wako Chemicals) based gel analysis was carried out to detect phosphorylated vimentin. Cos7 cells were infected with T. gondii RH or RH Δrop18 strains at a MOI of 3 for different periods of time (0, 1, 2, 6, 12, 24, 36, and 48h). Cells were then lysed with cell lysis buffer (TransGen Biotech, DE101) and the extracts were loaded on to 10% polyacrylamide gel (separating gel) containing 50μM Phos-tag acrylamide (Wako Chemicals, AAL-107). As vimentin expression levels changed with the time post T. gondii infection, the amount of cell lysate protein loaded for SDS-PAGE was adjusted to ensure a consistent level of total vimentin protein. After electrophoresis, Phos-tag acrylamide gels were washed three times with gentle shaking in transfer buffer containing 1mM EDTA for 10min and then incubated in transfer buffer containing 0.01% SDS without EDTA for 10 min, according to the manufacturer's protocol. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes using a standard immunoblotting protocol, and vimentin detected using a specific antibody (Supplementary Material ) 54 (link).
6
In Vitro Phosphorylation Assay for YeaG and AceA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For in vitro phosphorylation assays, 6 μg YeaG and 5 μg AceA were incubated at 37°C for 2 h in a reaction containing 50 mM HEPES pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 100 mM KCl, 10 mM ATP, and 0.1% triton X-100, with or without 10 mM malate. Reactions were started by adding ATP and stopped by adding sample buffer for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad) and boiling at 100°C. Samples were separated on 10% SDS-PAGE copolymerized with and without Phos-tag acrylamide (Fujifilm). To prepare SDS-PAGE copolymerized with Phos-tag acrylamide, 50 μM Phos-tag acrylamide and 100 μM MnCl2 in final concentration were added into resolving gel. Protein bands were revealed by Coomassie brilliant blue R-250 staining.
7
Phos-tag Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Phos-tagTM western blot, 8% acrylamide gel was mixed with 50 μmol/L Phos-tag™ acrylamide (Fujifilm, #AAL-107) and 100 μmol/L ZnCl2 (Sigma, #229997). After sample separation on a Zn2+-Phos-tag SDS/PAGE gel, the gel was incubated with gentle agitation in transfer buffer supplemented with 1 mM EDTA, 3 times for 10 min each, followed by washing with transfer buffer without EDTA for another 10 min. Proteins were transferred to nitrocellulose membranes using a wet-tank method and analyzed by western blotting. Ptbp1, His, and β-actin signals were detected simultaneously using enhanced chemiluminescence (ECL) western blotting detection reagents (Thermo Fisher Scientific).
8
Phosphorylation Analysis via Phos-tag SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine phospho-FoxO1, phospho-FoxO3a, and phospho-IKKβ protein levels, Phos-tag SDS-PAGE 37 (link) containing 50 mM Phos-tag acrylamide (Fujifilm) was used. Briefly, phosphorylated proteins and non-phosphorylated proteins were separated by Phos-tag SDS-PAGE. Phos-tag SDS-PAGE gel contained divalent Mn2+ ions, which trapped the phosphorylated proteins (such as p-FoxO1/p-FoxO3a/p-IKKβ) and increased their molecular weights. After electrophoresis, the Phos-tag gel was soaked in blotting buffer containing 1 mM EDTA (MilliporeSigma). The phosphorylated proteins and non-phosphorylated proteins on the Phos-tag gel were then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween® 20 (TBST) for 2 h, and then immunoblotted with anti-FoxO1, anti-FoxO3a, and anti-IKKβ antibodies to distinguish phosphorylated proteins from non-phosphorylated proteins.
9
Phosphorylated Protein Detection via Phos-tag SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect phosphorylated proteins, SDS-PAGE with or without 50 µM Phos-tag acrylamide (Wako chemicals) and 100 µM MnCl2 were used. After electrophoresis, Phos-tag acrylamide gels were washed using transfer buffer with 0.01% SDS and 1 mM EDTA for 10 min with gentle shaking and then replaced with transfer buffer with 0.01% SDS 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.
10
Phos-tag Acrylamide Affinity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phos-tag Acrylamide [11 (link)] is commercially available from Wako Pure Chemical Industries (Osaka, Japan). ATP and lithium potassium acetyl phosphate were purchased from Sigma-Aldrich (St. Louis, MO).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!