Recombinant DHPS-WT and DHPS variants were expressed in bacteria and purified using a GST protein tag. Both recombinant proteins were mixed with active ERK kinase (167820, United States Biological) and γ-32P-ATP (NEG002A100UC; PerkinElmer) in a kinase buffer (25 mM Tris [pH 7.5], 5 mM β-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2). These samples were incubated at 30 °C for 15 min. The reactions were stopped via boiling, and proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was dried and imaged using the PhosphorImager software program.
Neg002a100uc
Manufactured by PerkinElmer
Sourced in United States
The NEG002A100UC is a laboratory equipment product manufactured by PerkinElmer. It is a compact, high-performance device designed for use in various research and analytical applications. The core function of this product is to provide reliable and precise measurements, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t, by American Type Culture Collection (2 mentions)
G 25 column, by Cytiva (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Histone h3, by PerkinElmer (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Typhoon phosphorimager, by GE Healthcare (1 mentions)
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
γ tubulin, by Merck Group (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
Annexin 5 binding buffer, by BD (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
8 protocols using neg002a100uc
1
In vitro Kinase Assay of DHPS Variants
2
HER2 Kinase Activity Assay
3
Characterizing Tnk1 and Ack1 Signaling
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The UniProt IDs for human Tnk1 (isoform 2) and human Ack1 (isoform 1) are Q13470-2 and Q07912, respectively. HeLa and HEK293T cells were purchased from ATCC. Radiolabeled 32P-ATP was from PerkinElmer (NEG002A100UC). The following antibodies were used: phospho-STAT3 (CST9145P), STAT3 (CST4904S), phospho-AKT (CST4060S), AKT (CST9272S), FLAG (SigmaA8592), SBP (MilliporeMAB10764), Tnk1 (sc-390359), Tnk1 pY277 (MilliporeABS80), pTyr (Millipore05-321), GFP (sc-9996), His-tag (Biolegend906101), GST (MolecularProbes 83C1-3), PKM (sc-365684), Lamin A/C (sc-20681). (R)-9b inhibitor was purchased from Millipore Sigma (SML2073). Poly(Glu, Tyr) was purchased from Millipore Sigma (P0275). The peptides were synthesized and purified as described previously (21 ).
4
Synthesis and Labeling of DNA Substrates
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Single stranded nucleic acid strand was chemically synthesized from SigmaAldrich. DNA substrates were 5’ terminally labelled with T4 Polynucleotide Kinase (NEB, M0201S) and ATP-γ-32P (PerkinElmer, NEG002A100UC) following manufacturer’s protocol. Unincorporated free ATP was removed from reaction product using G-25 column (Cytiva, 27-5325-01). Equimolar concentration of oligos in annealing buffer (60 mM KCl, 1x PBS, and 0.2 mM MgCl2) were annealed by incubation at 95°C (5 min) then reduced to 25° C by step-wise intervals of 10° C (6 min/step). The dsDNA-88 substrate was produced by PCR amplification of R-loop-88-Fwd oligo with DNA Phusion polymerase.
5
In Vitro Kinase Assay for JAK2 and JAK3
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For in vitro kinase assay, 1 ng of human recombinant JAK3 active protein (Sigma, #14-629), 1 ng of human recombinant JAK2 active protein (Sigma, #14-640) and 2 μg of human recombinant Histone H3 (Sigma, #14-494) were used. Briefly, the kinase (JAK2 or JAK3) and the substrate (Histone H3) were re-suspended in a kinase buffer (20mM TRIS-HCl pH 7.5, 5 mM MgCl2, 5mM MnCl2, 1 μM cold ATP) containing 5 μCi γ-32P labelled ATP (Perkin Elmer, Waltham, MA, USA #NEG002A100UC) and were incubated at 37 °C for 30 min. The reaction was stopped by adding Laemmli SDS sample buffer and the samples were kept on ice immediately. For inhibition of JAK3, 100 nM Tofacitinib citrate was pre-incubated with the kinase for 30 min at room temperature. The kinase and the substrate were separated by running samples on 4–20% SDS-PAGE gel (BioRad, Hercules, CA, USA) followed by staining gel with Coomassie Brilliant blue (BioRad, #1610786). The gel was de-stained, dried, and kept in contact with storage phosphor image screen (GE Lifesciences, Marlborough, MA, USA) and the screen was visualized in a Typhoon Phosphorimager (GE Lifesciences).
6
Signaling Pathway Characterization Protocols
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[γ-32P]-ATP was purchased from PerkinElmer (NEG002A100UC). The synthetic peptides were purchased from Genemed Synthesis Inc and purified by semipreparative HPLC on a Vydac C18 column. HEK293T and DLD1 cells were purchased from the American Type Culture Collection. The following antibodies were used: FLAG (Sigma-Aldrich A8592), phospho-Stat3 (Tyr705) (CST9145), Stat3 (CST4904), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST4370), p44/42 MAPK (Erk1/2) (CST4695), γ-tubulin (Sigma-Aldrich T6557), ECL rabbit IgG (Cytiva NA9340), and ECL mouse IgG (Cytiva NA931). The molecular weights of proteins observed with these antibodies were consistent with the values in literature. In Western blotting experiments, we performed controls by omitting the primary antibody to test for potential background caused by the secondary antibodies. For Stat3, we confirmed results by using an alternative Stat3 antibody (F-2) (sc-8019).
7
Kinase Activity Assay for TNFR1 Domain
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Cells were treated with the indicated reagents for the indicated times and harvested and stained with Annexin V-FITC (BD Biosciences, 556547) and 7-AAD (eBioscience, 00-69936-50) in Annexin V binding buffer (BD Biosciences, 51-66121E) for 15 min. Post staining, the number of dead cells was measured using a BD Accuri C6 (BD Biosciences). Data are presented as mean ± standard deviation (S.D.), n = 3, with ns nonsignificance, *P < 0.05, **P < 0.01 and ***P < 0.001 at each point compared to the indicated graph with the two-sided Student's t test.
In vitro kinase assay 6xHis/TNFR1 DD constructs were subcloned into pcDNA3.1 and derived from 293 T cells. 6xHis/TNFR1 DD proteins were pulled-down using Ni-NTA agarose (Qiagen, #30210). Recombinant human EGFR protein (active) (Abcam, Cambridge, UK; ab268518) was procured from Abcam. Each protein was incorporated into TBS-based kinase buffer (Cell signalling, 9802 s) and incorporated 5 μM ATP (Affymetrix, 77241) and 2.5 μCi of [γ -P32]ATP (PerkinElmer, NEG002A100UC). The samples were incubated at 37 °C for 30 min. The reaction was terminated by incorporating sample buffer and incubating at 100 °C for 5 min. The reaction mixture was subjected to SDS-PAGE and radiography.
In vitro kinase assay 6xHis/TNFR1 DD constructs were subcloned into pcDNA3.1 and derived from 293 T cells. 6xHis/TNFR1 DD proteins were pulled-down using Ni-NTA agarose (Qiagen, #30210). Recombinant human EGFR protein (active) (Abcam, Cambridge, UK; ab268518) was procured from Abcam. Each protein was incorporated into TBS-based kinase buffer (Cell signalling, 9802 s) and incorporated 5 μM ATP (Affymetrix, 77241) and 2.5 μCi of [γ -P32]ATP (PerkinElmer, NEG002A100UC). The samples were incubated at 37 °C for 30 min. The reaction was terminated by incorporating sample buffer and incubating at 100 °C for 5 min. The reaction mixture was subjected to SDS-PAGE and radiography.
8
Rad53 Kinase Assay with Polymerase Epsilon
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