Superdex 200 increase 3.2 300 column

Manufactured by Cytiva

Sourced in Japan, United States

Superdex 200 Increase 3.2/300 is a prepacked size-exclusion chromatography column designed for fast and efficient separation of biomolecules. The column features a bed volume of 2.4 ml and a column dimension of 3.2 x 300 mm. It is suitable for analysis and purification of proteins, peptides, and other biomolecules with molecular weights ranging from 10,000 to 600,000 Da.

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (3 mentions) Superose 6 increase 3.2 300 column, by Cytiva (3 mentions) Lp 20ap, by Shimadzu (2 mentions) Ktamicro system, by GE Healthcare (1 mentions) Ultraufoil r 1.2 1 3 300 au mesh, by Quantifoil (1 mentions) Omnisec software, by Malvern Panalytical (1 mentions)

Spelling variants of this product

Superdex 200 (66 citations) Superdex 200 column (50 citations) Superdex 200 Increase (11 citations) Superdex 200 Increase 3.2/300 (6 citations) Superdex 200 Increase 3.2/300 GL column (2 citations) Superdex 200 Increase 3.2/300 size exclusion column (1 citations)

11 protocols using superdex 200 increase 3.2 300 column

Formation and Purification of ρ-Rof and ρ-rut Complexes

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For ρ-Rof complex formation, 41.8 μM ρ hexamer were mixed with a 10-fold molar excess of Rof in the absence or presence of 2.3 mM ADP-BeF₃ in reaction buffer and incubated for 15 min at 32°C. The mixture was applied to a Superdex 200 Increase 3.2/300 column (Cytiva) and fractions of the complex were pooled and concentrated. 3.8 μl of the purified complex (5.3 mg/ml) were applied to glow-discharged Quantifoil R1.2/1.3 holey carbon grids and plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher) set at 10 °C and 100 % humidity.
For ρ-rut RNA complex formation, 41.8 μM ρ hexamer were mixed with an equimolar amount of rut RNA and 2.3 mM ADP-BeF₃ in reaction buffer and incubated for 15 min at 32 °C. The mixture was applied to a Superose 6 Increase 3.2/300 column (Cytiva) and fractions of the complex were pooled and concentrated. Purified complex (3.8 mg/ml) was vitrified as above.

Said N., Finazzo M., Hilal T., Wang B., Selinger T.L., Gjorgjevikj D., Artsimovitch I, & Wahl M.C. (2023). Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation. bioRxiv.

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Protein Complex Assembly and Purification

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PSMD2 (15 µM) and MC1 (100 µM) were mixed together in 25 mM HEPES and 200 mM NaCl and allowed to bind for 10 min before adding 45 µM each of Fab 14 and Fab 8. The complex was treated with 0.0125% glutaraldehyde for 20 min at 25 °C before quenching with Tris (pH 7.5) to a final concentration of 300 mM. The treated complex was injected on a Superdex 200 Increase 3.2/300 column on an AKTAmicro (Cytiva). Concentration of the peak fraction was determined by absorbance at 280 nm.

Bashore C., Prakash S., Johnson M.C., Conrad R.J., Kekessie I.A., Scales S.J., Ishisoko N., Kleinheinz T., Liu P.S., Popovych N., Wecksler A.T., Zhou L., Tam C., Zilberleyb I., Srinivasan R., Blake R.A., Song A., Staben S.T., Zhang Y., Arnott D., Fairbrother W.J., Foster S.A., Wertz I.E., Ciferri C, & Dueber E.C. (2022). Targeted degradation via direct 26S proteasome recruitment. Nature Chemical Biology, 19(1), 55-63.

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Analytical Size Exclusion Chromatography Binding Assays

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All analytical size exclusion chromatography (SEC) binding assays were conducted on an ÄKTAmicro system (GE Healthcare) using a Superose 6 Increase 3.2/300 column (Cytiva, cat No. 29091598) equilibrated in 10 mM K-HEPES pH 8.0, 100 mM KCl, 0.5 mM TCEP, and 5% v/v glycerol. 2.5 μΜ apo-RNA Pol II was incubated on ice for 10 minutes with a 1.8 molar excess of APT or APT-subcomplexes in a total volume of 50 μl and spun (5 minutes at 21,130 x g) before injection. The eluted volume was monitored by A_260-280, collected in 50 μl fractions and analyzed by SDS-PAGE (NuPAGE™ 4-12%, Bis-Tris, Invitrogen) run in MES-SDS buffer at 190 V for 50 minutes.
Interaction studies between Glc7 and Ref2 were carried out by incubating 5 μΜ Glc7 with 10 μΜ Ref2 C-terminal truncations in 50 μl. Co-migration was assessed after injection on a Superdex 200 Increase 3.2/300 column (Cytiva, cat No. 28990946). The eluted volume was collected in 50 μl fractions and analyzed on SDS-PAGE (Bolt™ 4-12%, Bis-Tris, Invitrogen) run in MES-SDS buffer at 200 V for 35 minutes.

Carminati M., Rodríguez-Molina J.B., Manav M.C., Bellini D, & Passmore L.A. (2023). A direct interaction between CPF and RNA Pol II links RNA 3′ end processing to transcription. Molecular Cell, 83(24), 4461-4478.e13.

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Cryo-EM Structure Determination of ρ-Rof and ρ-rut Complexes

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For ρ-Rof complex formation, 41.8 µM ρ hexamer were mixed with a 10-fold molar excess of Rof in the absence or presence of 2.3 mM ADP-BeF₃ in reaction buffer and incubated for 15 min at 32 °C. The mixture was applied to a Superdex 200 Increase 3.2/300 column (Cytiva) and fractions of the complex were pooled and concentrated. 3.8 µl of the purified complex (5.3 mg/ml) were applied to glow-discharged Quantifoil R1.2/1.3 holey carbon grids and plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher) set at 10 °C and 100% humidity.
For ρ-rut RNA complex formation, 41.8 µM ρ hexamer were mixed with an equimolar amount of rut RNA (Supplementary Table 3) and 2.3 mM ADP-BeF₃ in reaction buffer and incubated for 15 min at 32 °C. The mixture was applied to a Superose 6 Increase 3.2/300 column (Cytiva) and fractions of the complex were pooled and concentrated. Purified complex (3.8 mg/ml) was vitrified as above.

Said N., Finazzo M., Hilal T., Wang B., Selinger T.L., Gjorgjevikj D., Artsimovitch I, & Wahl M.C. (2024). Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation. Nature Communications, 15, 3186.

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Analytical SEC of phosphorylated PINK1

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Analytical SEC was performed using a Superdex 200 Increase 3.2/300 column (Cytiva) equilibrated in SEC buffer. PhPINK1 (amino acids 115–575) D334A, D357A and monomeric phosphorylated PhPINK1 (amino acids 115–575, prepared as described above) were each, or in combination, diluted to 2 mg ml⁻¹ (per protein) in SEC buffer and incubatedovernight at 4 °C. 50 µl protein was loaded onto the column per run and eluted at 0.04 ml min⁻¹ flow rate.

Gan Z.Y., Callegari S., Cobbold S.A., Cotton T.R., Mlodzianoski M.J., Schubert A.F., Geoghegan N.D., Rogers K.L., Leis A., Dewson G., Glukhova A, & Komander D. (2021). Activation mechanism of PINK1. Nature, 602(7896), 328-335.

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ADAT2/3-tRNA Complex Formation

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For complex formation, ADAT2/3 heterodimer and tRNA were mixed and diluted in interaction buffer (20 mM Hepes pH 7.5; 50 mM NaCl; 2 mM MgCl2; 0.5 mM TCEP) to a final concentration of 32 µM and 35 µM, respectively. Excess tRNA was removed through size-exclusion chromatography on a Superdex 200 Increase 3.2/300 column (Cytiva). UltrAuFoil R 1.2/1.3 300 Au mesh (Quantifoil) grids were glow discharged with residual air for 30 seconds, on each side in a PELCO easiGlow device operated at 30 mA. Two microliters of sample were applied in each side of the grid before blotting for 3 s (blot force 0) and vitrified in liquid ethane using a Vitrobot MARK IV (Thermo Fisher Scientific) operated at 4 °C and 100% humidity.

Dolce L.G., Zimmer A.A., Tengo L., Weis F., Rubio M.A., Alfonzo J.D, & Kowalinski E. (2022). Structural basis for sequence-independent substrate selection by eukaryotic wobble base tRNA deaminase ADAT2/3. Nature Communications, 13, 6737.

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Molecular Weight Determination by SEC-MALS

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Size-exclusion chromatography (SEC) measurements were performed using a high-performance liquid chromatography (HPLC) system (LP-20AP, Shimadzu, Kyoto, Japan) and a Superdex 200 increase 3.2/300 column (Cytiva). The column was pre-equilibrated with 10 mM sodium phosphate (pH 6.0) and 150 mM NaCl, and samples of the wild-type and R371I mutant of Int8 were injected onto the column. The protein concentrations were 129 and 515 μM for the wild type and 138 and 550 μM for the R371I mutant. Molecular weight of the sample was determined on the HPLC system coupled to a Viscotek TDA 305 light scattering detector (Malvern Panalytical, Malvern, United Kingdom) as described previously (Chang et al., 2020 (link)). Bovine serum albumin (66.7 kDa) was used as a standard for instrument calibration. Data analysis for estimating molecular weights was performed using OmniSEC software (Malvern Panalytical). The error in molecular weight was calculated from duplicate or triplicate measurements.

Tashiro D., Suetaka S., Sato N., Ooka K., Kunihara T., Kudo H., Inatomi J., Hayashi Y, & Arai M. (2022). Intron-Encoded Domain of Herstatin, An Autoinhibitor of Human Epidermal Growth Factor Receptors, Is Intrinsically Disordered. Frontiers in Molecular Biosciences, 9, 862910.

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NDP52-FL SEC-SAXS Analysis Protocol

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Recombinantly expressed and purified NDP52-FL in SAXS buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM DTT) was used for SEC-SAXS experiments at a concentration of 5 mg/ml. NDP52-FL was analysed using a Superdex 200 increase 3.2/300 column, at a flow rate of 0.075 ml/min (Cytiva Life Sciences), using an Agilent 1200 HPLC system (Agilent LC). SEC-SAXS experiments were performed at the B21 Beamline, Diamond Light Source UK, by core facility staff. For SEC-SAXS analysis and envelope generation, ScÅtter software (Version J) was used in combination with the ATSAS package^{58 (link)}.

dos Santos Á., Rollins D.E., Hari-Gupta Y., McArthur H., Du M., Ru S.Y., Pidlisna K., Stranger A., Lorgat F., Lambert D., Brown I., Howland K., Aaron J., Wang L., Ellis P.J., Chew T.L., Martin-Fernandez M., Pyne A.L, & Toseland C.P. (2023). Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription. Nature Communications, 14, 2855.

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Size-exclusion Chromatography of Proteins

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For size-exclusion chromatography, proteins were mixed at a final concentration of 10 μM each in a volume of 50 μL 150 mM NaCl, 20 mM HEPES pH 7.5, 5 mM dithiothreitol. After a 5 h incubation at 4°C, samples were analyzed using an Superdex 200 Increase 3.2/300 column (Cytiva). Fractions were visualized using 10% SDS-PAGE polyacrylamide gels.

DAmico K.A., Stanton A.E., Shirkey J.D., Travis S.M., Jeffrey P.D, & Hughson F.M. (2023). Structure of a Membrane Tethering Complex Incorporating Multiple SNAREs. bioRxiv.

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Size Exclusion Chromatography of Protein

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20 μL of sample protein (2 mg/mL) in 20 mM Tris-HCl (pH 8.0) and 50 mM sodium chloride was loaded on a prepacked Superdex 200 Increase 3.2/300 column (Cytiva) prepared with 150 mM phosphate buffer (pH 7.3) and connected to Shimadzu Prominence high-performance liquid chromatography system with diode array detection (Shimadzu). The flow rate was set to 0.1 mL/min. Ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa) were used as MW reference standards.

Brangulis K., Akopjana I., Drunka L., Matisone S., Zelencova-Gopejenko D., Bhattacharya S., Bogans J, & Tars K. (2024). Members of the paralogous gene family 12 from the Lyme disease agent Borrelia burgdorferi are non-specific DNA-binding proteins. PLOS ONE, 19(4), e0296127.

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