All restriction and DNA modifying enzymes (T4 DNA ligase, Vent DNA polymerase, and calf intestine alkaline phosphatase) were from New England Biolabs (Ipswich, MA). Other chemicals were from sources recently described17 (link)23 (link).
Calf intestine alkaline phosphatase
Manufactured by New England Biolabs
Calf intestine alkaline phosphatase is an enzyme isolated from the intestinal mucosa of calves. It catalyzes the hydrolysis of phosphate groups from various substrates, including nucleotides, proteins, and alkaloids. The enzyme is commonly used in molecular biology applications as a tool for dephosphorylation reactions.
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Lab products found in correlation
Vent dna polymerase, by New England Biolabs (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Ribominus kit, by Thermo Fisher Scientific (1 mentions)
Rnase 3, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Thermo Fisher Scientific (1 mentions)
Carboplatin, by Merck Group (1 mentions)
Polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
Microspin g 50 column, by GE Healthcare (1 mentions)
Megascript high yield transcription kit, by Thermo Fisher Scientific (1 mentions)
Fastap, by Thermo Fisher Scientific (1 mentions)
Neb3 buffer, by New England Biolabs (1 mentions)
Coelenterazine h, by Nanolight Technology (1 mentions)
Flag m2 bead, by Merck Group (1 mentions)
Phosstop phosphatase inhibitor cocktail tablet, by Roche (1 mentions)
8 protocols using calf intestine alkaline phosphatase
1
DNA Manipulation Protocol Optimization
2
Isolation and Analysis of Membrane Fractions from Vertebrate Cells
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The total membrane (TM) and PM fractions of X. laevis oocytes (n = 10) and seabream ovarian follicles (n = 50) were isolated as described previously (Kamsteeg and Deen 2001 (link)). For the seabream follicles, the final pellet was resuspended in 1 × Laemmli sample buffer supplemented with 1 mM NaF, 1 mM Na3VO4, and protease inhibitors. In some experiments, whole protein extracts from seabream follicles were obtained by homogenization in 500 µl of radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with 1 mM NaF, 1 mM Na3VO4, protease inhibitors, and 80 U benzonase) using a glass douncer, and further incubation on ice for 15 min. The samples were then centrifuged at 14,000 × g at 4 °C for 1 min, and the supernatant was mixed with 2 × Laemmli sample buffer and heated at 95 °C for 10 min. In some cases, lysates were digested with calf intestine alkaline phosphatase (New England Biolabs Inc.; M0525) for 1 h at 37 °C before Laemmli sample buffer addition. Immunoblotting was subsequently carried out as previously described (Zapater et al. 2011 (link)).
3
Regulation of Gene Expression by DNMTi and Cytokine Signaling
4
In Vitro Transcript Synthesis and Labeling
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Transcripts used for in vitro assays (EMSA, RNA structure probing, 30S ribosome toeprinting) were synthesized with MEGAscript High Yield Transcription Kit (AM1333, Ambion). DNA templates with T7 promoter sequences were generated by PCR with oligonucleotides listed in Appendix Table S2 . RNA was isolated with phenol:chloroform:isopropanol (25:24:1), ethanol‐precipitated at −80°C and gel‐purified. For labelling, 20 pmol RNA was dephosphorylated with 10 units of calf intestine alkaline phosphatase (New England Biolabs) in a 20 μl reaction at 37°C for 1 h, followed by purification and precipitation, as above. The dephosphorylated RNA was 5′‐labelled with 3 μl of 32P‐γ‐ATP (10 Ci/l, 3,000 Ci/mmol) and 1 unit of polynucleotide kinase (Fermentas) for 1 h at 37°C in a 20 μl reaction. Unincorporated nucleotides were removed with Microspin G‐50 Columns (GE Healthcare), followed by purification of the labelled RNA on a denaturing polyacrylamide gel. Upon visualization of the labelled RNA with phosphorimager, the RNA band was excised from the gel and eluted with 0.1 M sodium acetate, 0.1% SDS, 10 mM EDTA at 4°C overnight, followed by phenol extraction and precipitation as before. Final concentrations were checked by NanoDrop 2000.
5
Dephosphorylation of Nuclear Pellets
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Nuclear pellets obtained from confluent MDCK monolayers were resuspended in NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol, pH 7.9; Cat. B7003S; New England Biolabs, Ipswich, MA). The nuclei were then sonicated and incubated for 60 min at 37°C with 20 U of calf intestine alkaline phosphatase (Cat. M0290S; New England Biolabs, Ipswich, MA). Samples were then processed for Western blot.
6
Dephosphorylation of Sf9-Tau by Alkaline Phosphatase
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Phosphorylated Sf9-Tau was incubated with 10 units of calf intestine alkaline phosphatase (New England Biolabs, catalog no. M0290) per 1 μg of protein for 24 h at 37 °C in NEB-3 buffer (New England Biolabs). The phosphatase was removed by high salt treatment (5 m NaCl at 70 °C), followed by centrifugation and dialysis to PBS for MALDI analysis. Dephosphorylation during TCSPC experiments was achieved by incubation of Sf9-Tau with 10 units/μg protein of Fast-AP (thermosensitive alkaline phosphate from ThermoFisher Scientific catalog no. EF0651) in PBS buffer at 25 °C.
7
Molecular Cloning Toolbox Protocol
8
Protein Immunoprecipitation and Phosphatase Assay
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Cells were solubilized for 1 h with lysis buffer containing 20 mM Tris, pH 7.4, 1% Triton X-100, 125 mM NaCl, 3 mM EDTA, 3 mM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1% SDS, 3.0 mM Na3VO4, 2.5 mM NaF, 2 mM tetra-Na pyrophosphate, and 25 mM β-glycerophosphate disodium salthydrate supplemented with protease inhibitor and PhosSTOP phosphatase inhibitor cocktail tablets (Roche). Cell lysates were passed through a syringe at least 15 times to disrupt the pellets, cleared at 16,100 × g for 10 min at 4°C and incubated with FLAG M2 beads (Sigma-Aldrich) overnight at 4°C. FLAG beads were washed once with lysis buffer, three times with 150 mM NaCl, 20 mM Tris, pH 8.0, 5 mM EDTA, 0.5% Triton X-100, 0.1% SDS, and 0.2% bovine serum albumin (BSA), three times with 500 mM NaCl, 20 mM Tris, pH 8.0, 0.5% Triton X-100, 0.2% BSA, and once with 50 mM Tris, pH 8.0. All of the washing buffers contained 2.5 mM NaF and 0.1 mM Na3VO4, except for the CIAP assay as indicated. FLAG-Gp78 immunoprecipitates were incubated with 50 U of calf intestine alkaline phosphatase (New England Biolabs) at 37°C for 1 h.
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