Ktamicro system

Manufactured by GE Healthcare

Sourced in Germany

The ÄKTAmicro system is a compact and automated chromatography system designed for fast and reliable protein purification. It provides precise control over flow rates, pressure, and fractionation, enabling efficient separation and purification of biomolecules.

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Lab products found in correlation

Sypro red staining, by Thermo Fisher Scientific (3 mentions) Snap surface alexa fluor 647, by New England Biolabs (2 mentions) Fluorescent image analyzer fla 3000, by Fujifilm (2 mentions) Minidawn treos, by Wyatt Technology (2 mentions) Kta pure, by Cytiva (1 mentions) Superdex 200 increase, by GE Healthcare (1 mentions) Bis tris gel, by Thermo Fisher Scientific (1 mentions) Sypro red, by Thermo Fisher Scientific (1 mentions) Superdex 75 10 300 gl, by Cytiva (1 mentions) Malls heleos dawn 2 detector, by Wyatt Technology (1 mentions) Zorbax 300 extend c18 column, by Agilent Technologies (1 mentions) Sep pak c18 cartridge, by Waters Corporation (1 mentions) Tmt 6 plex isobaric label reagent, by Thermo Fisher Scientific (1 mentions) Conalbumin, by GE Healthcare (1 mentions) Ovalbumin (ova), by GE Healthcare (1 mentions) Ribonuclease a, by GE Healthcare (1 mentions) Superdex 75 10 300 gl column, by GE Healthcare (1 mentions) Astra software suite, by Wyatt Technology (1 mentions) Superdex 200 5 150 column, by GE Healthcare (1 mentions) Rid 10a, by Shimadzu (1 mentions)

25 protocols using ktamicro system

Protein Purification Protocol

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Unless otherwise stated, all chemicals were purchased from Sigma (Sigma-Aldrich, Taufkirchen, Germany). FPLC-associated systems, ÄKTApure, and employed columns, were purchased from Cytiva (Freiburg, Germany). The ÄKTAmicro system was purchased from GE Healthcare (Freiburg, Germany).

Susemihl A., Nagel F., Grabarczyk P., Schmidt C.A, & Delcea M. (2021). Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain. Molecules, 26(24), 7576.

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Dynein-Lis1 Complex Stoichiometry

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2 µM Dyn^WB-M and 4 µM Lis1 were mixed and incubated for 10 min in DLB supplemented with 200 µM Mg-ATP/NaVO₄. Samples were fractionated on a Superdex 200 Increase 3.2/300 using an ÄKTAmicro system (GE Healthcare) that had been equilibrated in DLB buffer supplemented with 200 µM Mg-ATP/NaVO₄. Fractions were analyzed by SDS-PAGE on 4–12% Bis-Tris gels (Invitrogen) and visualized with SYPRO Red (Invitrogen). Peak fractions were then diluted so that both Dyn^WB-M and Lis1 were within the 50–500 ng range. Diluted samples were then re-analyzed via SDS-PAGE with an actin standard curve on the same gel to determine the absolute amount (ng) of each protein. Moles of each protein were calculated using 331,000 Da for Dyn^WB-M and 113,800 Da for dimeric Lis1. Molar ratios were then determined. We ensured that each protein binds SYPRO Red in a linear fashion by running titrations of Dyn^WB-M and Lis1 on SDS-PAGE gels and staining with SYPRO Red.

DeSantis M.E., Cianfrocco M.A., Htet Z.M., Tran P.T., Reck-Peterson S.L, & Leschziner A.E. (2017). Lis1 has two opposing modes of regulating cytoplasmic dynein. Cell, 170(6), 1197-1208.e12.

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Fluorescent Labeling of Motor Proteins

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SNAP_f-tagged motor proteins (~2.5 μM) were labeled in 100 μl reactions with 10 – 20 μM SNAP ligand (SNAP-Cell TMR-Star, SNAP-Surface Alexa Fluor 647 [NEB], or SNAP-conjugated oligonucleotide). Reactions were incubated at 4 °C for 2 hours. Proteins were purified from excess SNAP ligand by size-exclusion chromatography on a Superose 6 Increase 3.2/300 column using an ÄKTAmicro system (GE Healthcare), pre-equilibrated with gel filtration buffer (50 mM Tris [pH 7.5], 150 mM K-acetate, 2 mM Mg-acetate, 1 mM EGTA, 5% [v/v] glycerol, 1 mM DTT, 0.1 mM Mg-ATP). Fractions (100 μl) were analyzed by SDS-PAGE on 4-12% Tris-Bis gels with Sypro Red staining (Thermo Fisher Scientific), and imaged using an FLA-3000 fluorescent image analyzer (Fujifilm). Peak fractions were flash frozen under liquid nitrogen in single-use aliquots and stored at -80 °C.

Toropova K., Mladenov M, & Roberts A.J. (2017). Intraflagellar Transport Dynein is Autoinhibited by Trapping of its Mechanical and Track-Binding Elements. Nature structural & molecular biology, 24(5), 461-468.

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Protein Molar Mass Determination

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Absolute molar mass was calculated using the ÄKTAmicro system (GE Healthcare) coupled with a Dawn HELEOS II MALLS detector and an OptiLab T-rEX online refractive index detector (Wyatt Technology). Five hundred microliters of protein sample was injected onto the Superdex 200 10/300 GL HPLC size exclusion column (Cytiva) for DfrB-H5 and Superdex 75 10/300 GL (Cytiva) for DfrB1 at a flow rate of 0.4 ml min⁻¹. Bovine serum albumin was used for calibration.

Lemay-St-Denis C., Alejaldre L., Jemouai Z., Lafontaine K., St-Aubin M., Hitache K., Valikhani D., Weerasinghe N.W., Létourneau M., Thibodeaux C.J., Doucet N., Baron C., Copp J.N, & Pelletier J.N. (2023). A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype. Philosophical Transactions of the Royal Society B: Biological Sciences, 378(1871), 20220040.

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Quantitative Proteomic Analysis via TMT Labeling

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Lyophilized samples obtained from transferred 100 µl of the eluted peptide solution were resuspended in 100 µl of TEAB, and 20 µl of such peptide solution were labeled using a TMT 6-plex isobaric label reagent (Thermo Scientific, 90406). Each samples were labeled with 185 µg of TMTs according to the scheme presented in Fig. 2a and incubated overnight at room temperature before to be quenched by addition of 1-M Tris-HCL pH 7.5.
The labeled samples were combined, acidified, and desalted on Sep-Pak C18 cartridges (Waters, WAT051910). The desalted samples were dried, and resuspended in 5% acetonitrile, 5% ammonium hydroxide solution (pH 10.0), and then pre-fractionated into 80 fractions using a high pH reverse-phase Zorbax 300 Extend-C18 column (5 um, 4.6 × 250 mm, Agilent) on an ÄKTAmicro system (GE Healthcare). The pre-fractionated samples were concatenated into 20 injection fractions for each sample.

Bisteau X., Lee J., Srinivas V., Lee J.H., Niska-Blakie J., Tan G., Yap S.Y., Hom K.W., Wong C.K., Chae J., Wang L.C., Kim J., Rancati G., Sobota R.M., Tan C.S, & Kaldis P. (2020). The Greatwall kinase safeguards the genome integrity by affecting the kinome activity in mitosis. Oncogene, 39(44), 6816-6840.

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Protein Complex Fractionation by SEC

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For size-exclusion chromatography, indicated combinations of purified protein (100 picomoles each) were pre-incubated at a concentration of 1.1 μM for 10 min at 4°C. Samples were fractionated on a Superose 6 PC 3.2/30 column using an ÄKTAmicro system (GE Healthcare; Piscataway, NJ) that had been equilibrated with gel filtration buffer (50 mM Tris–HCl [pH 8.0], 150 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 5% glycerol, and 1 mM DTT). Fractions (50 μl) were analyzed by SDS-PAGE.

Roberts A.J., Goodman B.S, & Reck-Peterson S.L. (2014). Reconstitution of dynein transport to the microtubule plus end by kinesin. eLife, 3, e02641.

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Oligomerization State Analysis of NV10127-NV10129

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The oligomerization state of NV10127-NV10129 was analyzed by analytical size exclusion chromatography (SEC). Purified proteins (100 µM concentration in 50 mM TRIS 7.5, 50 mM KCl) were loaded on a Superdex 75 10/300 GL column (GE Healthcare) operated on an ÄKTAmicro system (GE Healthcare) connected to an ALIAS autosampler (Spark Holland). The system was operated at 25 °C with a flow rate of 0.5 mL/min of degassed buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl) and was calibrated with Conalbumin, Ovalbumin, Carbonic Anhydrase, Ribonuclease A and Aprotein from the GE Healthcare SEC Low-Molecular-Weight and High-Molecular-Weight calibration kits.

Semmelmann F., Hofferberth J., Ruther J, & Sterner R. (2019). Mapping key amino acid residues for the epimerase efficiency and stereospecificity of the sex pheromone biosynthetic short-chain dehydrogenases/reductases of Nasonia. Scientific Reports, 9, 330.

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Size Exclusion Chromatography-MALS Analysis

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SEC-MALS was performed at room temperature using an analytical Superdex200 5/150 column (GE Healthcare) equilibrated with GF buffer. SEC was performed with online static light scattering (miniDAWN TREOS, Wyatt Technology) and a differential refractive index (Shimadzu RID-10A) on an ÄKTAmicro system equipped with a triple wavelength UV detector (GE Healthcare). Data were analyzed using the ASTRA software suite (Wyatt Technology). The differential refractive index signal was combined with the light scattering to determine the molecular mass using standard protocols. A dn/dc of 0.178 was calculated for Olfm1 based on eight predicted N-linked glycans. Conalbumin was injected at 10 mg/ml as a control and calibration standard (for conalbumin, a dn/dc of 0.185 was used).

Pronker M.F., Bos T.G., Sharp T.H., Thies-Weesie D.M, & Janssen B.J. (2015). Olfactomedin-1 Has a V-shaped Disulfide-linked Tetrameric Structure. The Journal of Biological Chemistry, 290(24), 15092-15101.

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Protein Complex Formation Analysis

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Proteins (50 μM), alone or with an equimolar amount of binding partner, were incubated in SEC buffer for 30 min at 4 °C. 50 μl of sample were analyzed on Superdex 75 PC 3.2/30 or Superdex 200 Increase 3.2/300 size exclusion columns (GE Healthcare) using an ÄKTAmicro system (GE Healthcare) at 4 °C. The peak fractions were inspected by SDS-PAGE.

Ulrich A.K, & Wahl M.C. (2017). Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor. BMC Evolutionary Biology, 17, 91.

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Molecular Weight Analysis of Talin-Vinculin Complexes

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To test the molecular weight of the protein complexes, talin-FL and talin truncations as well as the mixtures of talin and vinculin (1:1 ratio) were prepared in buffer containing 20 mM HEPES, pH 7.5, 75 or 500 mM KCl, 0.5 mM EDTA at 3 mg/ml concentration and 20 μl were run on a Superdex 200 5/150 GL column on an ÄKTAmicro system (GE Healthcare) coupled to a Viscotek TDA302 detector (Malvern, Herrenberg, Germany) in the same buffer. Bovine serum albumin was used as a standard and the refractive index increment (dn/dc) was set to 0.180 ml/g for calculations. Data was analyzed using the OmniSEC 4.5 software (Malvern) and plotted with PRISM (GraphPad).

Dedden D., Schumacher S., Kelley C.F., Zacharias M., Biertümpfel C., Fässler R, & Mizuno N. (2019). The Architecture of Talin1 Reveals an Autoinhibition Mechanism. Cell, 179(1), 120-131.e13.

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