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Illustra nap 25 columns

Manufactured by GE Healthcare
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The Illustra™ NAP-25 columns are lab equipment designed for nucleic acid purification. The columns utilize a proprietary resin to facilitate the capture, washing, and elution of nucleic acids from various sample types.

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Lab products found in correlation

Mastercycler gradient pcr machine, by Eppendorf (1 mentions) His spintrap column, by GE Healthcare (1 mentions) Ms73 probe, by Bandelin (1 mentions) Infinite f200, by Tecan (1 mentions)

Close spelling variants of this product

Illustra NAP Columns Illustra NAP-25 NAPTM-25 columns NAP-25 column NAP column NAP-25

5 protocols using illustra nap 25 columns

1

Fumaric Acid-Alkyne Proteomics Analysis

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5 mg of HEK-293 proteome (2.5 mL, 2 mg/mL) was pre-treated with 1 mM fumaric acid (25 μL, 100 mM stock in DMSO) or DMSO for 3 hours prior to incubation with 100 μM FA-alkyne (25 μL, 10 mM stock in DMSO) for 15 hours. Proteomes were then desalted using Illustra™ NAP-25 columns (GE Healthcare # 17085201) to remove unreacted reagents. Labeled proteomes were enriched via Cu(I)-catalyzed [3 + 2] cycloaddition with biotin-azide as described above for chemoproteomic analysis. Following the final wash, enriched resin was collected on top of centrifugal filters (VWR, 82031–256). Proteins were eluted from resin via addition of 40 μL 1x SDS sample buffer, followed by boiling for 10 min at 95 °C. Following repetition of the elution step, both eluents were combined and 20 μL of the combined eluent was loaded onto a 4–12% SDS-PAGE gel and analyzed by western blotting.
Kulkarni R.A., Bak D.W., Wei D., Bergholtz S.E., Briney C.A., Shrimp J.H., Alpsoy A., Thorpe A.L., Bavari A.E., Crooks D.R., Levy M., Florens L., Washburn M.P., Frizzell N., Dykhuizen E.C., Weerapana E., Linehan W.M, & Meier J.L. (2019). A chemoproteomic portrait of the oncometabolite fumarate. Nature chemical biology, 15(4), 391-400.
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2

Oligonucleotide Synthesis and Purification

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Oligonucleotides were chemically synthesized in an Applied Biosystems DNA/RNA synthesizer, using the cyanoethyl phosphoramidite chemistry or purchased from Future Synthesis. All nucleotides and hexaethylene glycol phosphoramidites with 2΄-O-tertbutyldimethylsilyl were purchased from Glen Research, Azco, Proligo. The stilbene diether linker was synthesized using a custom synthesis service. Before the deprotection step, the click oligomers were treated with LiN3 (300eq) in dimethylformamide (DMF) for 2 h at 65°C. RNA oligomers were cleaved from the solid support using ammonium hydroxide/ethanol (3:1 v/v) at 55°C for 16 h. Deprotection was carried out with triethylamine trihydroflouride at 55°C for 3 h followed by the n-butanol precipitation for 1 h at 4°C. Oligomers were desalted on illustra NAP-25 columns (GE Healthcare) and purified by gel electrophoresis in denaturing conditions. In the case of the DNA oligomer it was cleaved and deprotected by ammonium hydroxide at 55°C for 16 h, desalted and purified by the denaturing gel electrophoresis.
Kiliszek A., Błaszczyk L., Kierzek R, & Rypniewski W. (2017). Stabilization of RNA hairpins using non-nucleotide linkers and circularization. Nucleic Acids Research, 45(10), e92.
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3

DNA Duplex Formation Protocol

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Single-stranded DNA sequences, purchased from Invitrogen, were re-suspended with nano-pure water to a final concentration of 1 mM and equimolar concentrations of complimentary strands were combined. Using an Eppendorf Mastercycler gradient PCR machine, samples were heated to 95 °C for 5 minutes. The samples were then cooled every 2 minutes in 5 °C increments to a final temperature of 20 °C. As trace amounts of tri-ethyl amine were detected, double-stranded DNA was further purified using Illustra NAP-25 columns (GE Healthcare) and DNA was eluted using nano-pure water. DNA samples were lyophilized overnight and re-suspended in nano-pure water to a final concentration of 15 mM.
Proudfoot A., Axelrod H.L., Geralt M., Fletterick R.J., Yumoto F., Deacon A.M., Elsliger M.A., Wilson I.A., Wüthrich K, & Serrano P. (2016). Dlx5 homedomain/DNA complex; Structure, binding and effect of mutations related to split-hand and foot malformation syndrome. Journal of molecular biology, 428(6), 1130-1141.
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4

Fumaric Acid-Alkyne Proteomics Analysis

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5 mg of HEK-293 proteome (2.5 mL, 2 mg/mL) was pre-treated with 1 mM fumaric acid (25 μL, 100 mM stock in DMSO) or DMSO for 3 hours prior to incubation with 100 μM FA-alkyne (25 μL, 10 mM stock in DMSO) for 15 hours. Proteomes were then desalted using Illustra™ NAP-25 columns (GE Healthcare # 17085201) to remove unreacted reagents. Labeled proteomes were enriched via Cu(I)-catalyzed [3 + 2] cycloaddition with biotin-azide as described above for chemoproteomic analysis. Following the final wash, enriched resin was collected on top of centrifugal filters (VWR, 82031–256). Proteins were eluted from resin via addition of 40 μL 1x SDS sample buffer, followed by boiling for 10 min at 95 °C. Following repetition of the elution step, both eluents were combined and 20 μL of the combined eluent was loaded onto a 4–12% SDS-PAGE gel and analyzed by western blotting.
Kulkarni R.A., Bak D.W., Wei D., Bergholtz S.E., Briney C.A., Shrimp J.H., Alpsoy A., Thorpe A.L., Bavari A.E., Crooks D.R., Levy M., Florens L., Washburn M.P., Frizzell N., Dykhuizen E.C., Weerapana E., Linehan W.M, & Meier J.L. (2019). A chemoproteomic portrait of the oncometabolite fumarate. Nature chemical biology, 15(4), 391-400.
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5

Bacterial Protein Expression and Purification

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For protein expression, bacteria were grown in the dark on plates with LB-agar supplemented with ampicillin for 3 days at room temperature. To measure crude extracts, we resuspended the bacteria in 20 mM MOPS pH 7.2, sonicated the suspension with an MS 73 probe (Bandelin) and centrifuged the sample with a tabletop centrifuge (Eppendorf). Protein extraction was performed with His Spin trap columns according to the manufacturer’s instructions (GE Healthcare). We resuspended the bacteria of one Petri dish in 2 ml binding buffer and sonicated with a MS 73 probe. Buffer exchange was performed with illustra NAP-25 columns (GE Healthcare). All measurements were performed in 20 mM MOPS (Sigma-Aldrich) with an Infinite F200 plate reader (Tecan).
Herud-Sikimić O., Stiel A.C., Kolb M., Shanmugaratnam S., Berendzen K.W., Feldhaus C., Höcker B, & Jürgens G. (2021). A biosensor for the direct visualization of auxin. Nature, 592(7856), 768-772.
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