Log In Sign up
The largest database of trusted experimental protocols

Calf intestinal alkaline phosphatase

Manufactured by Promega
Visit Supplier
Sourced in United States

Calf intestinal alkaline phosphatase is an enzyme extracted from calf intestine. It catalyzes the removal of 5' phosphate groups from DNA, RNA, and other molecules. The enzyme is commonly used in molecular biology applications.

Automatically generated - may contain errors

Lab products found in correlation

T4 polynucleotide kinase, by New England Biolabs (2 mentions) T4 dna ligase, by Promega (2 mentions) Complete edta free protease inhibitor, by Merck Group (1 mentions) Prism 6, by GraphPad (1 mentions) Glomax multi detection plate reader, by Promega (1 mentions) Hindiii endonuclease, by New England Biolabs (1 mentions) Bovine serum albumin (bsa), by Promega (1 mentions) T4 dna ligase enzyme, by Promega (1 mentions) Ultramer oligonucleotides, by Integrated DNA Technologies (1 mentions) T4 dna ligase enzyme, by New England Biolabs (1 mentions) Bbt atp, by Jena Biosciences (1 mentions) Dulbecco s phosphate buffered solution dpbs, by Merck Group (1 mentions) Image lab software, by Bio-Rad (1 mentions) Iblot system, by Thermo Fisher Scientific (1 mentions) Nupage novex 4 12 bis tris gradient gel, by Thermo Fisher Scientific (1 mentions) Immobilon reagent, by Merck Group (1 mentions) Anti gapdh, by Merck Group (1 mentions) Anti ps6k, by Cell Signaling Technology (1 mentions) Anti nkcc1, by Cell Signaling Technology (1 mentions) Gel doc xr system, by Bio-Rad (1 mentions)

Spelling variants of this product

Alkaline phosphatase (42 citations) Calf intestinal alkaline phosphatase (CIAP; (5 citations) Calf intestinal phosphatase (4 citations)

12 protocols using calf intestinal alkaline phosphatase

1

Phosphatase Treatment of Parasite Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
HFF monolayers grown in T25 flasks were infected with either GRA16-3xHA tagged parasites or untagged control parasites for 24 hours at an MOI of 2 under tachyzoite growth conditions. Adherent cultures were rinsed twice in cold PBS and then harvested in 500μL of cold RIPA buffer supplemented with cOmplete EDTA-free protease inhibitor (Sigma). Protein lysates were sonicated (30 seconds total, 20% amplitude, 1 second pulses) and incubated on ice for 30 minutes to allow for solubilization. Aliquots of sonicated and solubilized lysates containing approximately 2μg total protein were either treated with 100U calf intestinal alkaline phosphatase (Promega) in alkaline phosphatase buffer (50mM Tris-HCl pH 9.3, 1mM MgCl2, 0.1mM ZnCl2, 1mM spermidine) or mock treated with alkaline phosphatase buffer only and incubated in a 37C water bath for 1 hour. The reaction was then terminated by the addition of Laemmli buffer, and samples were analyzed by SDS-PAGE and immunoblotting as described below.
Mayoral J., Tomita T., Tu V., Aguilan J.T., Sidoli S, & Weiss L.M. (2020). Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export. PLoS Pathogens, 16(12), e1008771.
+ Open protocol
+ Expand
2

Enzyme Kinetics of Glycosyltransferase Fut8

Check if the same lab product or an alternative is used in the 5 most similar protocols
The relative enzyme activity of the substrates, A2-Asn, A2Asn-Fmoc, GlcNAc1Man5GlcNAc2-Asn, GlcNAc1Man5GlcNAc2-Asn-Fmoc, Man5GlcNAc2-Asn and Man5GlcNAc2-Asn-Fmoc, were determined by titrating with wild type Fut8 enzyme purified as reported earlier (27 ). Enzyme kinetics were performed using GDP-Glo™ Glycosyltransferase assay (Promega) for the substrates at a concentration range of 0–1mM (0–1.5 mM for Man5GlcNAc2-Asn) along with GDP-fucose (0.2 mM final concentration, pre-treated with Calf Intestinal alkaline-phosphatase (Promega)) as donor sugar (27 ). Reactions were carried out in a 10 μl reaction volume consisting of a universal buffer (200 mM each of Tris, MES, MOPS, pH 7.5) with the purified wild type enzyme at 37°C for 30 min. Reactions were stopped using 5 μl of GDP detection reagent and an equal volume of reaction mix in a polystyrene, white 384-well plate and incubating in dark for 1 h at room temperature. The luminescence values were measured using a GloMax Multi detection plate reader (Promega) and compared with a GDP standard curve to quantify the final released GDP product. The steady state parameters of KM, kcat, and kcat/KM values were determined using nonlinear curve fitting in GraphPad Prism 6 software.
Zhang R., Yang Q., Boruah B.M., Zong G., Li C., Chapla D., Yang J.Y., Moremen K.W, & Wang L.X. (2021). Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8). The Biochemical journal, 478(8), 1571-1583.
+ Open protocol
+ Expand
3

Custom Adapter Preparation for RNA-seq

Check if the same lab product or an alternative is used in the 5 most similar protocols
Custom acceptors and 5′ phosphorylated adapters (Table 1) were purchased from Integrated DNA Technologies (IDT) and gel purified using denaturing polyacrylamide gel electrophoresis (DPAGE). End-labeled adapters used at trace levels in adenylylation quantification experiments were dephosphorylated with calf intestinal alkaline phosphatase (Promega) and then re-5′-phosphorylated with [γ-32P]-ATP using T4 polynucleotide kinase according to the manufacturer's (New England Biolabs, NEB) instructions. Adapters 1T and 3 and acceptor 2 have previously been used in small RNA cDNA library construction (Williams et al. 2013 (link)).
Lama L, & Ryan K. (2016). Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I. RNA, 22(1), 155-161.
+ Open protocol
+ Expand
4

In situ DNA digestion and ligation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Samples for DNA accessibility mapping were prepared in the same way as BLISS samples, except that the in situ DSBs blunting step was substituted by an in situ DNA digestion step using 1 U μl–1 of HindIII endonuclease (NEB, catalogue number R3104) and incubating the samples for 18 h at 37 °C. HindIII cut sites were ligated with modified BLISS adapters carrying the HindIII complementary sticky end (see Supplementary Fig. 4h). To prevent in situ re-ligation of HindIII cut sites, the samples were incubated for 2 h at 37 °C in the presence of 0.015 U μl–1 of calf intestinal alkaline phosphatase (Promega, catalogue number M2825) before in situ ligation.
Yan W.X., Mirzazadeh R., Garnerone S., Scott D., Schneider M.W., Kallas T., Custodio J., Wernersson E., Li Y., Gao L., Federova Y., Zetsche B., Zhang F., Bienko M, & Crosetto N. (2017). BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nature Communications, 8, 15058.
+ Open protocol
+ Expand
5

Cloning of Wheat RabD2a Gene

Check if the same lab product or an alternative is used in the 5 most similar protocols
Bovine serum albumin, calf intestinal alkaline phosphatase, T4 DNA ligase enzyme and BamHI and BglII restriction enzymes were acquired from Promega. A 270-bp section of the CDS of wheat RabD2a gene Ta.54382 was obtained by PCR using primers with BamHI and BglII restriction sites added to their 5′ ends (Table 3, Figure S7). The PCR product was purified and cloned into the vector pHMW-Adh-Nos (Nemeth et al., 2010 (link); Figure S1). Digestion and ligation of insert and vector were performed using the supplier's instructions. Negative controls used were no enzyme digest (for both insert and vector digests), no enzyme ligation, no vector ligation and no insert ligation. Competent DH5-α E. coli cells were used for transformation. E. coli cells were grown in LB+ampicillin medium (Per plate: 10 mL sterile LB medium mixed with 10 μL 100 mg/mL ampicillin).
Tyler A.M., Bhandari D.G., Poole M., Napier J.A., Jones H.D., Lu C, & Lycett G.W. (2014). Gluten quality of bread wheat is associated with activity of RabD GTPases. Plant Biotechnology Journal, 13(2), 163-176.
+ Open protocol
+ Expand
6

Coligo Circularization: DNA Ultramer Oligonucleotides

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
5′ phosphorylated synthetic linear DNA Ultramer® oligonucleotides were purchased from Integrated DNA Technologies. Coligo secondary structures were predicted using the mfold program.14 (link) Circularization of coligo precursors discontinuous in regions predicted to be ss (122, 19aTAR, 221, 19am3, luc-1, luc-2, Dcr3, 15a-b, 19a, mut2A, mut2B, 19a-rb, 19a-rb-122L, 19a-rb-122LS, 19a-rb5, 19a-rb7, 19a-rb9) was performed using TS2126 RNA ligase enzyme as previously described.21 (link) 5′ phosphorylated nicked dumbbell or dumbbell-like linear DNA Ultramers (19anbm19, 19anbm23, 19anbm29, 19anbm34, 19anb-BR1, 19anb-BR2, 19anb-BR3, 19anb-BR4, 19anb-BR5) were circularized using T4 DNA ligase enzyme (New England BioLabs, NEB, M0202S) and purified as described below. Unphosphorylated coligo precursors used in Figures 5A-C were made by dephosphorylating the purchased Ultramer oligo using Calf intestinal alkaline phosphatase (Promega, M182A) according to the manufacturer’s instructions. Similarly, 5′ [γ-32P]-ATP labeling of dephosphorylated linear Ultramer for Figures 5D and 5E was done using T4 Polynucleotide kinase (NEB, M0201S) according to the manufacturer’s instructions.
Lama L., Seidl C.I, & Ryan K. (2014). New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III. Transcription, 5(2), e27913.
+ Open protocol
+ Expand
7

DENV NS5 Polymerase Inhibitor Assay

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
The cell-based experiments were performed as previously described33 (link). Briefly, the Huh-7 cells were transfected with 0.5 µg of p(+)RLuc-(–)DV-UTRΔC-FLuc and DENV NS5 expression vector pcDNA-NS5-Myc followed by compound treatment for 3 days. The RLuc and FLuc activities were analysed by Dual-Glo Luciferase Assay System37 (link). The enzyme-based fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) was performed as previously described33 (link). Briefly, the template was amplified from the cDNA of DENV-2 minus strand 3′-UTR and its RNA was synthesised by the T7 Megascript kit38 (link). The 100 nM DENV NS5 protein was incubated with 1, 5 or 10 µM test compound, 100 nM (−) 3′-UTR RNA, 20 µM CTP, GTP, UTP and 20 µM BBT-ATP (Jena Bioscience) in a total volume of 30 µL in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM KCl, 1 mM MgCl2, 0.3 mM MnCl2, 0.001% Triton X-100 and 10 µM cysteine) at 25 °C for 60 min. The 30 µL of 2.5× stop buffer (200 mM NaCl, 25 mM MgCl2, 1.5 M diethanolamine, pH 10) with 25 nM calf intestinal alkaline phosphatase (Promega) was added to the reaction after 60-min incubation. The fluorescence signal was measured at excitation wavelength of 422 nm and emission wavelength of 566 nm, respectively.
Pelliccia S., Wu Y.H., Coluccia A., La Regina G., Tseng C.K., Famiglini V., Masci D., Hiscott J., Lee J.C, & Silvestri R. (2017). Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities. Journal of Enzyme Inhibition and Medicinal Chemistry, 32(1), 1091-1101.
+ Open protocol
+ Expand
8

Porcine Atrioventricular Valve Leaflet Culture

Check if the same lab product or an alternative is used in the 5 most similar protocols
Hearts were obtained from healthy, non-pregnant female pigs immediately after slaughter at a local abattoir (Holifield Farms, Covington, GA, USA). The AV leaflets were immediately excised, thoroughly rinsed, and stored in sterile Dulbecco’s phosphate-buffered solution (dPBS; Sigma, St. Louis, MO, USA) at 4°C for transport to the laboratory. Upon arrival at the laboratory, five pieces (each 5 mm2) were cut aseptically from the basal region of each leaflet in a laminar flow hood. Each leaflet sample was placed in a separate well of a 12-well tissue culture plate with 2.4 ml of Dulbecco’s modified Eagle medium (DMEM; Mediatech, Herndon, VA, USA) containing penicillin and streptomycin for eight days in a 5% CO2 incubator at 37°C, with media changes every two days. The DMEM contains 1.8 mM calcium and 0.8 mM phosphate, and the phosphate concentration was increased by adding NaH2PO4. For some studies, the valves were devitalized prior to culture by repetitive freezing and thawing (five to six cycles). The following additives were used when indicated: inorganic pyrophosphatase (from bakers’ yeast; Sigma Diagnostics, St. Louis, MO, USA), 1000 Units/ml stock solution in Hanks buffered salt solution; calf intestinal alkaline phosphatase (Promega, Madison, WI, USA), 1000 units/ml suspension; and etidronic acid (TCI America, Portland, OR, USA), 1 mM stock solution in distilled water.
Rathan S., Yoganathan A.P, & O’Neill W.C. (2014). The Role of Inorganic Pyrophosphate in Aortic Valve Calcification. The Journal of heart valve disease, 23(4), 387-394.
+ Open protocol
+ Expand
9

Western Blot Analysis of OXSR1 and Downstream Targets

Check if the same lab product or an alternative is used in the 5 most similar protocols
Equal amounts of total cell extracts were run under denaturing conditions using NuPAGE Novex 4–12% Bis-Tris gradient gels (Life Technologies). Proteins were transferred onto nitrocellulose membranes using the iBlot system (Life Technologies), with a standard (P0, 8 min) protocol. Nitrocellulose was blocked for 60 min in 5% w/w non-fat dried milk (Marvel) in Tris buffered saline/0.05% Tween-20 (TBST). Primary antibodies used were: Anti-OXSR1 (Abcam, ab224248), 1/1000; anti- Phospho-OXSR1 (Abcam, ab138655), 1/1000; anti-S6K (Cell signaling, 2708), 1/2000; anti-P-S6K (Cell signaling, 9205), 1/1000; anti-NKCC1 (Cell Signaling, 14581), 1/500; anti-GAPDH (Sigma, G9545), 1/2000; HRP-conjugated secondary antibody was antirabbit (Sigma-Aldrich, A6154), 1/5000. Chemiluminescence detection was performed using Immobilon reagent (Millipore) and the Gel-Doc™ XR+ system (Bio-Rad). Quantification was performed using the ImageJ gel quantification plugin or Image Lab software (Biorad). To better identify phopho-OXSR1 band, one of the replicate samples was treated with Calf intestinal alkaline phosphatase (Promega, M182A) for 60 min at 37 °C.
Stangherlin A., Watson J.L., Wong D.C., Barbiero S., Zeng A., Seinkmane E., Chew S.P., Beale A.D., Hayter E.A., Guna A., Inglis A.J., Putker M., Bartolami E., Matile S., Lequeux N., Pons T., Day J., van Ooijen G., Voorhees R.M., Bechtold D.A., Derivery E., Edgar R.S., Newham P, & O’Neill J.S. (2021). Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology. Nature Communications, 12, 6035.
+ Open protocol
+ Expand
10

Mutagenesis of Arabidopsis MET1 Enzyme

Check if the same lab product or an alternative is used in the 5 most similar protocols
DNA fragments with compatible ends were ligated in a reaction incubated for 17 h at 4 oC using 1 U of T4 DNA ligase (Promega). De-phosphorylation was carried out using calf intestinal alkaline phosphatase (Promega) according to the manufacturer’s instructions. 5’ overhangs produced after amplicon assembly were filled by PCR using the Phusion high-fidelity PCR kit (Finnzymes). Arabidopsis transformation was carried out by floral dip [24 (link)].
The MET1 cDNA [25 (link)] was cut from p-GEM T easy (Promega) using EcoRI and was subsequently ligated into pGreen II 0179 35S-NOS, which contains a single EcoRI site in the polylinker region between the promoter and terminator. To remove the catalytic function from MET1 we followed the strategy documented by Hsieh et al [26 (link)] and exchanged the cysteine residue in the active site loop region in MET1 GGPPCQGFSGMNRFN by a serine residue. Site-directed mutagenesis and subsequent assembly-PCR were used to mutate the cysteine codon (TGT) to a serine codon (TCT) within the MET1 coding sequence.
Brocklehurst S., Watson M., Carr I.M., Out S., Heidmann I, & Meyer P. (2018). Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1). PLoS ONE, 13(2), e0192170.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!