Phos-tag gels were prepared with gel containing 40 μM of Phos-tag Acrylamide (Wako) and 80 μM MnCl2. Samples were prepared in EDTA-free RIPA and treated with calf intestinal phosphatase (ThermoFisher) at 1 unit per microgram of protein for 30 min at 37 °C for 30 min before adding loading buffer.
Calf intestinal phosphatase
Manufactured by Thermo Fisher Scientific
Sourced in United States
Calf intestinal phosphatase is an enzyme derived from calf intestines. It catalyzes the hydrolysis of various phosphate esters and functions to remove phosphate groups from molecules.
Automatically generated - may contain errors
Lab products found in correlation
Ribonuclease t1, by Thermo Fisher Scientific (2 mentions)
Phos tag acrylamide, by Fujifilm (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Monarch dna gel extraction kit, by New England Biolabs (1 mentions)
S1 nuclease, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Tobacco acid pyrophosphatase, by Thermo Fisher Scientific (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
γ 32p atp, by Hartmann Analytic (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
Fla 5100, by Fujifilm (1 mentions)
6 protocols using calf intestinal phosphatase
1
Phosphatase Assay with Phos-tag Gel
2
Linearized Plasmid Transfection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reporter assay has been previously described (20 (link),21 (link)). pGL3-Control (Promega) plasmid was digested with HindIIII (Thermo Scientific) to create a double-strand break between the promoter and firefly luciferase coding sequence. Digested plasmid was then treated with calf intestinal phosphatase (Thermo Scientific) to mitigate spontaneous re-ligation. Linearized plasmid was confirmed by electrophoresis and isolated using the Monarch DNA Gel Extraction Kit (New England Biolabs). Cells were plated at a density of 25 000 per well in a 96-well plate. After 24 h, each well was transfected with 50 ng of either linearized or uncut plasmid as well as 10 pg of Renilla luciferase vector as a transfection efficiency control using FuGENE 6. Luciferase activity was measured 24 h after transfection using the Dual Luciferase Reporter Assay System (Promega) and normalized to the Renilla luciferase signal. Data are reported as a percentage of the normalized firefly luciferase activity from cells transfected with uncut pGL3-Control plasmid. All assays were done in triplicate.
3
Structure Probing of cnfY 5'-UTR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Structure probing of the 5’-UTR and 80 nt of cnfY was performed with in vitro transcribed RNA using pBO4465 or pBO4466 as template. The in vitro transcribed RNA was purified and dephosphorylated using CIP enzyme (Calf intestinal phosphatase, Thermo Scientific, Waltham, USA). The RNA was labeled with [32P] at the 5′ end as described elsewhere [31 (link)]. Partial digestions of radiolabeled RNA with ribonuclease T1 (0.0025 U) (Thermo Scientific, Waltham, USA) and T2 (0.056 U) (MoBiTec, Göttingen, Germany) were performed according to [32 (link)] at 25, 37, and 42°C. For digestion with RNase T1 and RNase T2 a 5 x TN buffer (100 mM Tris acetate, pH 7, 500 mM NaCl) was used. An alkaline hydrolysis ladder was prepared as described before [31 (link)]. The T1-ladder was generated by using 30000 cpm labeled RNA. The RNA was heated with 1 μl sequencing buffer (provided with RNase T1) at 90°C. Afterwards, the RNA was incubated with the enzyme at 37°C for 5 min.
4
Characterization of the TPP Riboswitch
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TPP riboswitch, the P3a::4U* RNA and its variants rep (A6C) and derep (G12A/C23U) were obtained by in vitro transcription. The RNA was purified and dephosphorylated with CIP (Calf intestinal phosphatase, Thermo Scientific, Waltham, USA). RNA was labeled at the 5′ end as described (31 (link)). Partial digestions with ribonuclease T1 (0.00125 U) (Ambion, Austin, USA) and nuclease S1 (0.125 U) (Thermo Scientific, Waltham, USA) were performed according to (19 (link)) at indicated temperatures in absence or presence of 100 μM TPP. For digestion with RNase T1, 5x TN buffer (100 mM Tris acetate, pH 7, 500 mM NaCl) was used. Digestion with nuclease S1 was performed using the supplied 5× reaction buffer. An alkaline hydrolysis ladder (31 (link)) and a T1-ladder were prepared. For the T1-ladder 30,000 cpm labeled RNA was incubated with 1 μl sequencing buffer (provided with RNase T1) at 90°C followed by incubation with the enzyme at 45°C for 5 min.
5
Determination of Viral RNA 5' and 3' UTR Sequences
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Viral RNA was isolated using a QIAamp viral RNA minikit (Qiagen), and cDNA libraries were prepared using Superscript III (Invitrogen). Sanger sequencing was performed on PCR templates generated using Phusion High-Fidelity DNA polymerase (New England BioLabs) and analyzed using Sequencher (Gene Codes) and Lasergene (DNAStar) software.
The 5′ and 3′ UTR sequences were determined as previously described (53 (link)). Briefly, viral RNA was isolated and treated with calf intestinal phosphatase (Ambion) for 1 h at 37°C to remove terminal phosphates of uncapped RNAs. Following a phenol-chloroform extraction and isopropanol precipitation, the RNA was treated with tobacco acid pyrophosphatase (Ambion) for 1 h at 37°C. After another phenol-chloroform extraction and isopropanol precipitation, the RNA was incubated with T4 RNA ligase I (Ambion) for 1 h at 37°C and then overnight at 4°C, to ligate the 5′ and 3′ ends. cDNA (Superscript III; Invitrogen) was made and used to generate an amplicon containing both the 5′ and 3′ UTR sequences, which was Sanger sequenced.
The 5′ and 3′ UTR sequences were determined as previously described (53 (link)). Briefly, viral RNA was isolated and treated with calf intestinal phosphatase (Ambion) for 1 h at 37°C to remove terminal phosphates of uncapped RNAs. Following a phenol-chloroform extraction and isopropanol precipitation, the RNA was treated with tobacco acid pyrophosphatase (Ambion) for 1 h at 37°C. After another phenol-chloroform extraction and isopropanol precipitation, the RNA was incubated with T4 RNA ligase I (Ambion) for 1 h at 37°C and then overnight at 4°C, to ligate the 5′ and 3′ ends. cDNA (Superscript III; Invitrogen) was made and used to generate an amplicon containing both the 5′ and 3′ UTR sequences, which was Sanger sequenced.
6
Purification and Analysis of piRNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
piRNAs with a length of 28 to 35 nt from the testes and ovaries were purified from 5–8 µg of low-molecular-weight RNA by excision of the corresponding gel slices from SYBR-gold-stained, 15% denaturing polyacrylamide gels. The piRNAs were recovered from the excised gel slices by elution with 0.3 M sodium acetate and 1 mM EDTA buffer. Approximately 150 ng of the piRNA fraction from both gonads was 5′-end-labeled using T4 polynucleotide kinase (Ambion) in the presence of [γ-32P] ATP (Hartmann Analytic), followed by dephosphorylation using calf intestinal phosphatase (Ambion). The labeled piRNAs with and without alkaline phosphatase treatment were separated on a 12% polyacrylamide-urea gel. Fujifilm FLA-5100 was used for radioactive imaging analysis.
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!