Bacterial strains of E. coli (NCTC 12241, 11560, 13351) and S. aureus (NCTC 12981, 12973) were obtained from Pro-Lab Diagnostics (UK). Nitrocefin was obtained from Merck Millipore (UK). Chlorophenol red-β-D-galactopyranoside (CPRG), 5-bromo-6-chloro-3-indolyl phosphate p-toluidine salt (BCIP, magenta phosphate) or magenta phosphate, β-galactosidase, β-lactamase and rAPid alkaline phosphatase were all acquired from Sigma-Aldrich (UK). HMRZ-86 was purchased from Bio-Rad Laboratories (UK). Filter paper (Whatman grade 4) was bought from GE Healthcare Life Sciences (UK). Mueller-Hinton broth and Buffered Peptone Water broth were purchased from Oxoid Ltd, UK. Pasteurised cow's milk was purchased from local stores (Hull, UK).
Rapid alkaline phosphatase
Manufactured by Merck Group
Sourced in United Kingdom
RAPid alkaline phosphatase is a laboratory reagent used to detect and quantify the presence of the enzyme alkaline phosphatase in biological samples. It provides a rapid and reliable method for measuring alkaline phosphatase activity.
Automatically generated - may contain errors
Lab products found in correlation
T4 polynucleotide kinase pnk, by New England Biolabs (2 mentions)
Rnasin ribonuclease inhibitor, by Merck Group (2 mentions)
Micro bio spin p 30 columns, by Bio-Rad (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
Nitrocefin, by Merck Group (1 mentions)
Buffered peptone water broth, by Thermo Fisher Scientific (1 mentions)
β galactosidase, by Merck Group (1 mentions)
Mueller hinton broth (mhb), by Thermo Fisher Scientific (1 mentions)
β lactamase, by Merck Group (1 mentions)
4 protocols using rapid alkaline phosphatase
1
Antimicrobial Activity Evaluation
2
Phosphopeptide Enrichment and Quantification
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Phosphopeptides enriched from yeast and HeLa cells as described above were each split 50:50. One-half was dephosphorylated using rAPid alkaline phosphatase (Sigma-Aldrich; 1 μl per 2 mg protein starting material) at 37 °C 750 r.p.m. shaking overnight, while the other half was mock treated using water. Both samples were incubated for 10 min at 85 °C to inactivate the phosphatase, and then purified on C18 StageTips33 (link). Yeast phosphopeptides were mixed at 0.1%, 1%, 10%, 50%, 90%, 99%, and 99.9% into dephosphorylated peptides at 99.9%, 99%, 90%, 50%, 10%, 1%, and 0.1%, with the total amount of peptides per condition corresponding to Ti-IMAC enrichment from 200 μg yeast starting material. Mixtures were then added into a 1:1 HeLa mixture of phosphopeptides and dephosphorylated peptides, each corresponding to a total amount of peptides per condition corresponding to Ti-IMAC enrichment from 100 μg HeLa starting material. Samples were then measured with the DDA and optimized DIA method as described below.
3
RNA 5' Dephosphorylation and Radiolabeling
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RNA was dephosphorylated using rApid Alkaline Phosphatase (Millipore) to convert 5′-triphosphate to 5′-monophosphate ends. To this end, 100 pmol RNA was incubated in a 20 µL reaction containing 2 µL rApid Alkaline Phosphatase buffer, 2 µL rApid Alkaline Phosphatase, 1 µL RNasin Ribonuclease Inhibitor (Sigma-Aldrich), and incubated for 30 min at 37°C. The enzyme was heat-inactivated for 2 min at 75°C. The RNA was subsequently 5′ end-labeled with [γ32-P] ATP (PerkinElmer) using T4 polynucleotide kinase (PNK) (New England Biolabs). This was done by adding 1 µL 100 mM MgCl2, 4 µL PNK Buffer, 2 µL of 5 mCi [γ32-P] ATP, and 2 µL of PNK in a total volume of 40 µL. The reaction mix was incubated for 30 min at 37°C and purified using Micro Bio P-30 spin columns (Bio-Rad).
4
5' RNA Labeling and Degradation Assay
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4 µg RNA was resuspended in 40 µL 100 mM NaCl, 10 mM MgCl2, 50 mM Tris pH 7.5, 1 mM DTT and re-folded at 90°C for 3 minutes then 20°C for 5 minutes. 3 µL recombinant RppH (0.5 µg/µL stock) was added and the samples were split into two 20 µL reactions (-/+ exoribonuclease). 1.5 µL of the recombinant Xrn1 (0.8 µg/µL stock) was added where indicated. All reactions were incubated for 1.5 hrs at 30°C using a thermocycler. The degradation reactions were resolved on a 7 M urea 10% denaturing polyacrylamide gel and stained with ethidium bromide.
32 P-5¢-end labeling of RNA RNA was dephosphorylated using rApid Alkaline phosphatase (Millipore) to convert 5¢-triphosphate to 5¢monophosphate ends. To this end, 100 pmol RNA was incubated in a 20 µl reaction containing 2 µl rApid Alkaline phosphatase buffer, 2 µl rApid Alkaline phosphatase, 1 µl RNasin Ribonuclease Inhibitor (Sigma-Aldrich), and incubated for 30 mins at 37°C. The enzyme was heat-inactivated for 2 mins at 75°C. The RNA was subsequently 5¢-end labeled with [γ32-P] ATP (PerkinElmer) using T4 polynucleotide kinase (PNK) (New England Biolabs). This was done by adding 1 µl 100mM MgCl2, 4 µl PNK Buffer, 2 µl of 5 mCi [γ32-P] ATP, and 2 µl of PNK in a total volume of 40 µl. The reaction mix was incubated for 30 mins at 37°C and purified using Micro-Bio P-30 spin columns (Bio-Rad).
32 P-5¢-end labeling of RNA RNA was dephosphorylated using rApid Alkaline phosphatase (Millipore) to convert 5¢-triphosphate to 5¢monophosphate ends. To this end, 100 pmol RNA was incubated in a 20 µl reaction containing 2 µl rApid Alkaline phosphatase buffer, 2 µl rApid Alkaline phosphatase, 1 µl RNasin Ribonuclease Inhibitor (Sigma-Aldrich), and incubated for 30 mins at 37°C. The enzyme was heat-inactivated for 2 mins at 75°C. The RNA was subsequently 5¢-end labeled with [γ32-P] ATP (PerkinElmer) using T4 polynucleotide kinase (PNK) (New England Biolabs). This was done by adding 1 µl 100mM MgCl2, 4 µl PNK Buffer, 2 µl of 5 mCi [γ32-P] ATP, and 2 µl of PNK in a total volume of 40 µl. The reaction mix was incubated for 30 mins at 37°C and purified using Micro-Bio P-30 spin columns (Bio-Rad).
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