In vitro gel-shift assays were performed with 1 µg GST-TRF1 wt or mutant (T330A, S344A; T248A) and 32P-labeled ds(TTAGGG)7 telomeric probe. Briefly, DNA probes were prepared by annealing the two oligonucleotides, sense (5′-GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGCCCCTC-3′) and antisense (5′-GAGGGGCCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCC-3′), end-labeled with [γ-32P]ATP (Amersham Biosciences) and T4-polynucleotide kinase (New England BioLabs) and purified by free nucleotide removal spin column (Qiagen). Labeled-DNA probes were incubated with 1 μg of purified TRF1 protein for 20 min at room temperature in a 30-μl reaction containing 20 mm HEPES-KOH, pH 7.9, 150 mm KCl, 5% (v/v) glycerol, 4% (w/v) Ficoll, 1 mm EDTA, 0.1 mm MgCl2, 0.5 mm dithiothreitol, and 20 μg of bovine serum albumin, and poly(dI-dC) (1 μg) as the nonspecific competitor (Amersham Biosciences). For supershift analysis, recombinant TRF1 was preincubated with anti-TRF1 antibody (rat homemade) before EMSA. The DNA-protein complexes were resolved on a 5% non-denaturing polyacrylamide (29:1 acrylamide:bisacrylamide) gel with 0.5× Tris-borate-EDTA, TBE (Sigma) as running buffer. Gels were dried under a vacuum at 80 °C and auto radiographed.
γ 32p atp
Manufactured by Cytiva
Sourced in United States, United Kingdom, Italy, Germany
[γ-32P]ATP is a radioactive compound containing the phosphorus-32 isotope. It is a commonly used reagent in molecular biology and biochemistry laboratories for various applications, including nucleic acid labeling, protein phosphorylation studies, and enzyme activity assays.
Automatically generated - may contain errors
Lab products found in correlation
T4 polynucleotide kinase, by New England Biolabs (7 mentions)
Poly di dc, by Roche (4 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Chemiluminescence detection kit, by Cytiva (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Mts solution, by Promega (2 mentions)
Cnbr sepharose 4b, by Cytiva (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Genescreen plus membrane, by PerkinElmer (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
T4 polynucleotide kinase, by Cytiva (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Poly di dc, by Cytiva (1 mentions)
Liquid scintillation counter, by Beckman Coulter (1 mentions)
G 25 spin columns, by GE Healthcare (1 mentions)
Phosphorimager screen, by Bio-Rad (1 mentions)
Imagequant, by Bio-Rad (1 mentions)
54 protocols using γ 32p atp
1
In Vitro Telomeric DNA Binding Assay
2
DYRK1A Kinase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the IVK assays, immunocomplexes or GST-fusion proteins expressed in bacteria (see Supplementary Methods) were incubated for 20 min at 30 °C, in the presence or absence of GST-DYRK1A, in 20 µl of phosphorylation buffer (25 mM Hepes [pH 7.4], 5 mM MgCl2, 5 mM MnCl2, 0.5 mM DTT) that contained 50 µM ATP and 2.5 µCi [γ-32P]-ATP (3,000 Ci/mmol, Amersham Biosciences). The incorporation of 32P was determined by SDS-PAGE and exposing the dried gel to film.
DYRK1A kinase activity was assessed using the DYRKtide peptide as the substrate40 (link). Briefly, DYRK1A-immunocomplexes were incubated for 20 min at 30 °C in 20 µl of phosphorylation buffer including 200 µM DYRKtide and 2.5 µCi [γ-32P]-ATP (3,000 Ci/mmol). Aliquots of the reaction were dotted onto P81 Whatman phosphocellulose paper in triplicate, washed extensively with 5% orthophosphoric acid and counted in a liquid scintillation counter (Beckman Coulter).
DYRK1A kinase activity was assessed using the DYRKtide peptide as the substrate40 (link). Briefly, DYRK1A-immunocomplexes were incubated for 20 min at 30 °C in 20 µl of phosphorylation buffer including 200 µM DYRKtide and 2.5 µCi [γ-32P]-ATP (3,000 Ci/mmol). Aliquots of the reaction were dotted onto P81 Whatman phosphocellulose paper in triplicate, washed extensively with 5% orthophosphoric acid and counted in a liquid scintillation counter (Beckman Coulter).
3
Electrophoretic Mobility Shift Assay for Transcription Factor Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear extracts from HB1.F3 cells were prepared as described earlier (Kim et al., 2013 (link)). Sense and antisense oligonucleotides were annealed and then end-labeled with [γ-32P]ATP (Amersham) and T4 polynucleotide kinase. Labeled probes were purified on 19% non-denaturing polyacrylamide gels. The DNA–protein binding reaction was performed in a final volume of 20 μl reaction buffer containing 10 mM Tris (pH 7.6), 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl2, 5% glycerol, and 250 μg of poly(dI–dC) per milliliter. Nuclear extract (20 μg of protein) was added to the reaction buffer in the absence or presence of unlabeled competitor DNA and pre-incubated for 15 min on ice. Radioisotope-labeled probes (50,000 cpm) were added, and the mixture was incubated for a further 30 min at room temperature. To resolve DNA–protein complexes, electrophoresis was performed on a 5% non-denaturing polyacrylamide gel. Gels were fixed, dried, and visualized by autoradiography. The oligonucleotides employed were as follows (only sense strands presented):
NMU-NurRE, 5'-GTTCCTCACCTTTCAAAGGGAGGTCAAATA-3';
NMU-mtNurRE, 5'-GTTCCCTGTT TTTCAAAAACAG GTCAAATA-3'; and
G0S2-NBRE2, 5'-CATCACTGACCTTTGCAATT-3'.
NMU-NurRE, 5'-GTTCCTCACCTTTCAAAGGGAGGTCAAATA-3';
NMU-mtNurRE, 5'-GTTCC
G0S2-NBRE2, 5'-CATCACTGACCTTTGCAATT-3'.
4
Telomeric DNA Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Telomeric duplex DNA 5′-GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGCCCCTC-3′ and antisense (5′-GAGGGGCCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCC-3′ was end-labeled with [γ-32P] ATP (Amersham Biosciences) and T4-polynucleotide kinase (New England BioLabs) and purified from free nucleotides through G25 spin columns (GE Healthcare). Binding was carried out by incubating 0.5 ng of labeled DNA with 1 μg of unmodified or PARP1 covalently PARylated TRF1 (as above described) in 15 μl of a reaction mix of 20-mM Hepes (pH 7.9), 100-mM NaCl, 50-mM KCl, 1-mM MgCl2, 0.1-mM ethylenediaminetetraacetic acid (EDTA), 1 mM DTT, 5% (v/v) glycerol, 0.5 mg/ml of BSA and 0.1% (v/v) NP-40. Samples were incubated at 4 °C for 90 min and then run on native 4.5% polyacrylamide gels. Gels were dried and exposed to PhosphorImager screens and acquired using ImageQuant (Bio-Rad), and the signals were measured using ImageQuant software (Quality One; Bio-Rad).
5
RNA Oligonucleotide Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA oligonucleotides were synthesized and 5′-end labeled using T4 polynucleotide kinase (NEB, Beverly, MA, USA) and [γ-32P]ATP (Amersham). All labeled oligonucleotides were gel purified on 12% denaturing gels prior to use. Electrophoretic mobility shift assays (EMSA) were carried out as described previously (24 (link)) with minor modifications. A total of 5 ng of each probe (∼20 000 cpm) was mixed with 30 μg of K562 S100 extract, The incubation was in 20 μl of binding buffer (10 mM Tris-HCl [pH 7.4], 150 mM KCl, 1.5 mM MgCl2 and 0.5 mM dithiothreitol) at room temperature for 20 min. One microliter of heparin (50 mg/ml) was added to each reaction mixture for 10 min prior to loading. Samples were resolved on a non-denaturing 5% polyacrylamide gel.
6
Cdk2 kinase activity assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates (200 μg) in lysis buffer (1% Triton X-100, 150 mM NaCl, 100 mM KCl, 20 mM HEPES pH 7.9, 10 mM EDTA, 1 mM sodium orthovanadate [SOV], 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 μM PMSF) were incubated with anti-Cdk2 antibody (1 μg/mL) overnight at 4 °C. Following this, 20 μL Protein G-agarose (Santa Cruz Biotechnology) was added, and the lysates were incubated for 2 h at 4 °C. Protein G-agarose conjugates were pelleted by centrifugation, washed thrice with lysis buffer and twice with kinase buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, 1 μM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin), and then resuspended in 30 μL kinase buffer. Samples were incubated at 37 °C for 30 min with 20 μM ATP, 5 μCi [γ-32P]-ATP (Amersham), and 1 μg histone H1 (US Biological, Salem, MA, USA). The reaction was stopped by adding 5X sample loading buffer (60 mM Tris, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM β-mercaptoethanol, and 0.1% bromophenol blue). The kinase reaction mixtures were separated by SDS-PAGE on 12% gels, and phosphorylated substrates were detected by autoradiography.
7
FAM20c Kinase Phosphorylation of VWF
8
Purification and Characterization of LexA Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Over-expression and purification of the LexA protein was performed as previously described (Da Re et al., 2009 (link)). The EMSA probes were obtained by PCR using oligonucleotides 8 and 9 (probe PintI1, 270bp, Supplementary Table S1 ) or 10 and 11 (probe PintI2, 233bp, Supplementary Table S1 ) amplified from pAT674 or genomic DNA of isolate Dak 0898/12-14, respectively. They were end-labeled with [γ32P]ATP (Amersham, Saclay, France) using T4 DNA polynucleotide kinase (Promega, Charbonnières, France). The EMSA experiments were performed as previously described (Da Re et al., 2009 (link)) using various amounts of purified LexA, 40 ng of the radiolabelled DNA probe PintI1 or PintI2 in the binding mixtures, and 630 ng of unlabelled probe for competition experiments (around 15.75-fold excess).
9
Mapping Phytochemical Signaling Cascades
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
6,7,4'-THIF was obtained from Chromadex™ (Irvine, CA, USA), and Dulbecco’s modified eagle medium (DMEM) and MMP-1 antibody were purchased from Thermo Fisher Scientific (San José, CA, USA). Medium 199 (M199) and daidzein were purchased from Sigma-Aldrich (St. Louis, MO, USA), and fetal bovine serum (FBS) was purchased from Gemini Bio-Products (Calabasas, CA, USA). CNBr-Sepharose 4B, [γ-32P]-ATP and the chemiluminescence detection kit were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). The protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). MTS solution was from Promega (Madison, WI, USA). Penicillin/streptomycin was purchased from Invitrogen (Grand Island, NY, USA). Primary antibodies recognizing phosphorylated MEK (Ser217/221), total MEK, phosphorylated SEK1/MKK4 (MKK4, Ser257/Thr261), phosphorylated MKK3 (Ser189)/6 (Ser207), total MKK3, phosphorylated p38 (Tyr180/182), total p38 and PKCα were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against phosphorylated-ERKs (Tyr204), total ERKs, ERK2, total MKK4, phosphorylated JNK (Thr183/Tyr185), total JNK, JNK2 and phosphorylated PKCα (Ser657) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Active PKCα and PKCδ proteins were purchased from Millipore (Bedford, MA, USA).
10
Nuclease and Phosphatase Assays
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!