Illustra microspin g 50 column, by GE Healthcare - Product details

1

Radiolabeling of DNA Markers for Molecular Weight Analysis

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Standard molecular weight markers were prepared by dephosphorylating 17 μg λ DNA-HindIII Digest (New England Biolabs N3012S) with 10 U Antarctic Phosphatase (New England Biolabs M0289S) in total volume of 40 μL for 1 h at 37°C. The phosphatase was then inactivated by incubation at 80°C for 10 min. Subsequently, 6.8 μg of dephosphorylated DNA was labeled with γ-[³²P]-ATP using 40 units of T4 Polynucleotide Kinase (New England Biolabs M0201S) for 1 h at 37°C, in a total reaction volume of 40 μl. Unincorporated γ-[³²P]-ATP was removed using Illustra MicroSpin G-50 columns (GE Healthcare) and 5 mM EDTA was added to the recovered sample.
For Figure 1D, end-labeled 3189 bp plasmid was prepared by digesting 4 μg plasmid DNA with 2 μL SmaI (Roche) in 1X CutSmart Buffer (New England Biolabs) in a 40 μL final reaction volume for 2 h at 25°C. The linearized plasmid was then column purified using the High Pure PCR Product Purification Kit (Roche) and then dephosphorylated and end-labeled with γ-[³²P]-ATP, as described for standard molecular weight markers. Other labeled markers in Figure 1D were generated by PCR amplification using oligonucleotides 7272 – 7275 (see Table S2) and pTDK13 plasmid template. In each case, 50 μL PCR reactions were assembled in the presence of 33 nM α-[32P]-dCTP and the PCR products were purified over Illustra MicroSpin G-50 columns (GE Healthcare).

Deegan T.D., Baxter J., Ortiz Bazán M.Á., Yeeles J.T, & Labib K.P. (2019). Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes. Molecular Cell, 74(2), 231-244.e9.

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2

ADP-Ribosylation of Proteins by ARTD Enzymes

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To obtain ADP-ribosylated ARTD1, H3 and H2B tails, and HMGB1, recombinant proteins were incubated in reaction buffer RB (50 mM Tris-HCl pH 7.4, 4 mM MgCl₂ and 250 μM dithiothreitol (DTT)) at a ratio 1:3 (ARTD1 to histone tail) with 100 nM [³²P]NAD⁺ (Perkin Elmer) and 200 nM of double-stranded annealed 40 bp long oligomer (5ʹ-TGCGACAACGATGAGATTGCCACTACTTGAACCAGTGCGG-3ʹ) for 15 min at 37 °C. ADP-ribosylation was stopped by adding 10 μM PJ34.
Automodification of ARTD8 was carried out in RB with 150 nM [³²P]NAD⁺ and 10 μM cold NAD⁺ for 1 h at 37 °C. The reaction was stopped by filtering through an Illustra MicroSpin G-50 column (GE Healthcare) according to the manufacturer’s manual.
ADP-ribosylation of actin was performed as described earlier^{45 (link)}. Briefly, 2 μg β/γ actin (Cytoskeleton Inc.) was incubated with 50 ng CDTa in the presence of 100 nM [³²P]NAD⁺, 150 μM cold NAD⁺ and reaction buffer (5 mM HEPES, pH 7.5, 0.1 mM CaCl₂, 0.5 mM NaAc, 0.1 mM ATP) at 37 °C. The reaction was stopped by filtering through a G50 column.

Abplanalp J., Leutert M., Frugier E., Nowak K., Feurer R., Kato J., Kistemaker H.V., Filippov D.V., Moss J., Caflisch A, & Hottiger M.O. (2017). Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase. Nature Communications, 8, 2055.

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3

Radioactive Labeling of DNA Probes

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The oligonucleotide sequences used in this study are listed in Table 1, and the DNA probes used in this study are listed in Figure 3. The labelling of the DNA probes was performed as previously described [38] . In brief, a 0.5 µM stock of the appropriate oligonucleotide was isotopically labelled at the 5′-end with 0.034 µM of [γ-³²P] ATP (10 mCi/ml) (Perkin Elmer, Waltham, MA, USA) and 10 units of T4 polynucleotide kinase (New England Biolabs). The oligonucleotide was then purified on an Illustra MicroSpin G-50 column (GE Healthcare, Piscataway, NJ, USA) following the manufacturer's protocol. Each DNA substrate was annealed by heating 0.5 µM of the ³²P-labeled oligonucleotide with a 0.4 µM solution of the appropriate unlabelled oligonucleotides from 10 uM stock solutions in 10 mM Tris-HCl (pH 8.0) containing 100 mM NaCl for 3 minutes at 100°C and allowing the reaction to cool to room temperature in 2.5–3 hours. The annealed DNA substrates were drop-dialyzed against 4 mL of 1× TE (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA)) for 10 minutes using a 25 mm 0.025 µm nitrocellulose membrane disc (Millipore, Billerica, MA, USA), and the Holliday Junction DNA substrate was purified using the crush and soak method as previously described [39] (link).

Tjokro N.O., Rocco C.J., Priyadarshini R., Davey M.E, & Goodman S.D. (2014). A Biochemical Analysis of the Interaction of Porphyromonas gingivalis HU PG0121 Protein with DNA. PLoS ONE, 9(3), e93266.

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4

Radioactive Oligonucleotide Labeling

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20 pmol of an oligo was incubated for 30 min at 37°C with γ-[³²P]-ATP and 10 U of T4 polynucleotide kinase (Thermo Scientific) in (50 mM Tris–HCl [pH 7.6], 10 mM MgCl₂, 5 mM DTT, 0.1 mM spermidine) in a total volume of 20 μl. After incubation for 10 min at 80°C, free γ-[³²P]-ATP was removed using an Illustra MicroSpin G-50 column (GE Healthcare).

Sterk M., Romilly C, & Wagner E.G. (2018). Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites. Nucleic Acids Research, 46(8), 4188-4199.

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5

Synthesis and Characterization of TAR RNA

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TAR RNAs (WT, Δbulge and Δloop) were synthesized in in-vitro transcription reactions with the Riboprobe system (Promega).^{35 (link)} Transcripts were treated with 2 U of DNase I (Promega), extracted with a phenol:chloroform mixture and purified over an Illustra MicroSpin G50 column (GE Healthcare). Gel-mobility reactions (12 μl final volume) were carried out in binding buffer (50 mM Tris, pH 7.4, 0.5 mM EGTA, 150 mM NaCl, 2% glycerol, 0.2% Tween 20, 0.5 mM DTT, 90 mM ZnSO4, 0.005% BSA and 100 μM ATP) and contained 2×10⁴ cpm TAR probes/reaction and the indicated concentrations of Tat and recombinant SMYD5 (Active Motif). Reactions were incubated for 30 min at 30 °C and separated on a pre-run 4% Tris-glycine gel. The gels were dried and exposed to Amersham Hyperfilm (GE Healthcare) overnight.

Boehm D., Lam V., Schnolzer M, & Ott M. (2023). The lysine methyltransferase SMYD5 amplifies HIV-1 transcription and is post-transcriptionally upregulated by Tat and USP11. Cell reports, 42(3), 112234.

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6

Synthesis and Characterization of TAR RNA

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TAR RNAs (WT, Δbulge and Δloop) were synthesized in in-vitro transcription reactions with the Riboprobe system (Promega).^{35 (link)} Transcripts were treated with 2 U of DNase I (Promega), extracted with a phenol:chloroform mixture and purified over an Illustra MicroSpin G50 column (GE Healthcare). Gel-mobility reactions (12 μl final volume) were carried out in binding buffer (50 mM Tris, pH 7.4, 0.5 mM EGTA, 150 mM NaCl, 2% glycerol, 0.2% Tween 20, 0.5 mM DTT, 90 mM ZnSO4, 0.005% BSA and 100 μM ATP) and contained 2×10⁴ cpm TAR probes/reaction and the indicated concentrations of Tat and recombinant SMYD5 (Active Motif). Reactions were incubated for 30 min at 30 °C and separated on a pre-run 4% Tris-glycine gel. The gels were dried and exposed to Amersham Hyperfilm (GE Healthcare) overnight.

Zamir I., Dawson J., Lavinsky R.M., Glass C.K., Rosenfeld M.G, & Lazar M.A. (1997). Cloning and characterization of a corepressor and potential component of the nuclear hormone receptor repression complex. Proceedings of the National Academy of Sciences of the United States of America, 94(26), 14400-14405.

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7

Radioactive DNA Oligonucleotide Labeling Protocol

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DNA decoys in DNase/RNase free water were annealed by heating to 95 °C in a heating block for 5 minutes, followed by slow cooling to room temperature. To a microcentrifuge tube (1.7 mL) was added the annealed DNA decoy (50 pmol) and T4 polynucleotide kinase (PNK) buffer (5 μL of a 10X solution, Thermo Scientific). DNase/RNase-free H₂O was added to yield a final volume of 40 μL. The reaction tube was placed into a shielded rack then [γ-³²P]-ATP (5 μL; 6,000 Ci/mmol, Perkin Elmer) was added. PNK was diluted in DNase/RNase-free H₂O (1:10) then added to the reaction (5 μL). The reaction was briefly mixed, centrifuged (to remove any material from cap), and then placed in a 37 °C heat block for 30 minutes. Heating to 70 °C for 30 minutes in the second heat block was then used to inactivate the kinase. The radioactive reaction mixture was transferred to an Illustra MicroSpin G-50 column (GE Healthcare, prepared according to vendor instructions) and centrifuged at 1500 rpm for 20 seconds to yield ³²P-labeled oligonucleotides. The radioactivity of the oligonucleotides were quantified (counts/min/μL) by transferring an aliquot to an Eppendorf tube followed by analysis on a Beckman LS 6500 multi-purpose scintillation counter (dry counting).

Struntz N.B, & Harki D.A. (2016). Catch and Release DNA Decoys: Capture and Photochemical Dissociation of NF-κB Transcription Factors. ACS chemical biology, 11(6), 1631-1638.

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8

In Vitro ADP-Ribosylation Assays

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In vitro ADP-ribosylation assays were performed based on previously described methods^{65 (link),66 (link)}. For auto-ADP-ribosylation assays recombinant ARTD1 (10 pmol) was incubated in reaction buffer (RB; 50 mM Tris-HCl pH 7.4, 4 mM MgCl₂, and 250 μM dithiothreitol (DTT)) with 100 μM NAD⁺ and 200 nM of double-stranded annealed 40 bp long oligomer (5′-TGCGACAACGATGAGATTGCCACTACTTGAACCAGTGCGG-3′, 5′-CCGCACTGGTTCAAGTAGTGGCAATCTCATCGTTGTCGCA-3′) for 15 min at 37 °C. Recombinant ARTD8cat was incubated in RB buffer with either 100 μM NAD⁺ or 200 nM [³²P] NAD⁺ (Perkin Elmer) for 30 min at 37 °C. These reactions were stopped via the addition of SDS buffer or by filtering through an Illustra MicroSpin G-50 column (GE Healthcare), according to the manufacturer’s protocol. De-ADP-ribosylation assays were performed in RB buffer. For de-modification of ARTD1, the auto-modified recombinant proteins were incubated with 10 pmol PARG for 30 min at 37 °C. The hydrolysis activities of WT Af1521 or eAf1521 were tested by incubating auto-modified ARTD8cat with either 10 pmol of recombinant WT Af1521, or eAf1521 for 2 h at 4 °C or 37 °C. De-modification of auto-modified ARTD8cat was then visualized by SDS–PAGE and autoradiography. For generation of PAR chains, poly-ADP-ribosylated ARTD1 was digested using proteinase K at 42 °C for 1 h.

Nowak K., Rosenthal F., Karlberg T., Bütepage M., Thorsell A.G., Dreier B., Grossmann J., Sobek J., Imhof R., Lüscher B., Schüler H., Plückthun A., Leslie Pedrioli D.M, & Hottiger M.O. (2020). Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins. Nature Communications, 11, 5199.

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9

UVDE Digestion of UV-induced Photoproducts

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The following oligos were used for UVDE digestion of UV-induced TA photoproducts:
Forward Primer 5′-GCGTGTGCACGTATATATATACGCGCGTGTG-3′ and
Reverse Primer 5′Biotin-CACACGCGCGTATATATATACGTGCACACGC-5′.
Annealed oligos were labeled with [γ³²P]ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB, M0201L). The labeled primers were purified using Illustra Microspin G-50 column (GE healthcare) and 40 μL of the purified, labeled DNA was spotted onto glass coverslips as four spots (10 μl each) for each dose and exposed to UVC light using UV StratalinkerTM 1800. The spots were recovered from the coverslip and ethanol precipitated. The damaged DNA samples were digested with UVDE enzyme in a reaction buffer (pH 6.5) containing HEPES (20mM), NaCl (100mM), and MnCl₂ (1mM) at 55þC for 1 hour. Digested DNA was then ethanol precipitated, washed and dissolved in 5 μL of deionized water. Formamide was added to the samples to a final concentration of 50% and heated at 80þC for 5 minutes and loaded on to a pre-run 15% denaturing polyacrylamide urea gel. The gel was run for 2 hours and 10 minutes. The gel was exposed to phosphor screen and the radioactivity signal was imaged using a Typhoon scanner (GE Healthcare). The TA lesion band intensity was quantified using ImageQuant TL software.

Laughery M.F., Brown A.J., Bohm K.A., Sivapragasam S., Morris H.S., Tchmola M., Washington A.D., Mitchell D., Mather S., Malc E.P., Mieczkowski P.A., Roberts S.A, & Wyrick J.J. (2020). Atypical UV Photoproducts Induce Non-canonical Mutation Classes Associated with Driver Mutations in Melanoma. Cell reports, 33(7), 108401.

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10

UVDE Digestion of UV-induced Photoproducts

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The following oligos were used for UVDE digestion of UV-induced TA photoproducts:
Forward Primer 5′-GCGTGTGCACGTATATATATACGCGCGTGTG-3′ and
Reverse Primer 5′Biotin-CACACGCGCGTATATATATACGTGCACACGC-5′.
Annealed oligos were labeled with [γ³²P]ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB, M0201L). The labeled primers were purified using Illustra Microspin G-50 column (GE healthcare) and 40 μL of the purified, labeled DNA was spotted onto glass coverslips as four spots (10 μl each) for each dose and exposed to UVC light using UV StratalinkerTM 1800. The spots were recovered from the coverslip and ethanol precipitated. The damaged DNA samples were digested with UVDE enzyme in a reaction buffer (pH 6.5) containing HEPES (20mM), NaCl (100mM), and MnCl₂ (1mM) at 55þC for 1 hour. Digested DNA was then ethanol precipitated, washed and dissolved in 5 μL of deionized water. Formamide was added to the samples to a final concentration of 50% and heated at 80þC for 5 minutes and loaded on to a pre-run 15% denaturing polyacrylamide urea gel. The gel was run for 2 hours and 10 minutes. The gel was exposed to phosphor screen and the radioactivity signal was imaged using a Typhoon scanner (GE Healthcare). The TA lesion band intensity was quantified using ImageQuant TL software.

Laughery M.F., Brown A.J., Bohm K.A., Sivapragasam S., Morris H.S., Tchmola M., Washington A.D., Mitchell D., Mather S., Malc E.P., Mieczkowski P.A., Roberts S.A, & Wyrick J.J. (2020). Atypical UV Photoproducts Induce Non-canonical Mutation Classes Associated with Driver Mutations in Melanoma. Cell reports, 33(7), 108401.

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Illustra microspin g 50 column

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36 protocols using illustra microspin g 50 column

Radiolabeling of DNA Markers for Molecular Weight Analysis

ADP-Ribosylation of Proteins by ARTD Enzymes

Radioactive Labeling of DNA Probes

Radioactive Oligonucleotide Labeling

Synthesis and Characterization of TAR RNA

Synthesis and Characterization of TAR RNA

Radioactive DNA Oligonucleotide Labeling Protocol

In Vitro ADP-Ribosylation Assays

UVDE Digestion of UV-induced Photoproducts

UVDE Digestion of UV-induced Photoproducts

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