Hiload 26 600 superdex 200 pg column

Manufactured by GE Healthcare

Sourced in Germany, United Kingdom, United States

The HiLoad 26/600 Superdex 200 pg column is a size-exclusion chromatography column designed for protein purification. It features a bed volume of 320 mL and a column dimension of 26 mm x 600 mm. The column packing material is a proprietary composite of cross-linked agarose and dextran, providing high resolution and desalting capabilities.

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Superdex 200 (746 citations) Superdex 200 column (565 citations) HiLoad Superdex 200 (24 citations) Superdex 200 pg (22 citations) HiLoad 26/600 Superdex 200 column (13 citations) HiLoad 26/600 Superdex 200 (8 citations) Superdex 200 26/600 column (7 citations) HiLoad Superdex 200 pg (7 citations) Superdex 200 26/600 (5 citations) Superdex 200 pg 26/600 column (3 citations)

25 protocols using hiload 26 600 superdex 200 pg column

Purification and Binding Analysis of SNX9 and β2-HAD

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GST-SNX9 in a pGEX-KG vector and His6x-β2-HAD in a pET vector (gift of Dr. Linton Traub, University of Pittsburgh, Pittsburgh, PA) were expressed in BL21(DE3) and then affinity purified using glutathione Sepharose 4B beads (ABT) and a HisTrap HP column (GE Healthcare). The affinity-purified GST-SNX9 was thrombin-digested to remove GST. The resulting SNX9 and β2-HAD proteins were separately applied to a HiLoad 26/600 Superdex 200 pg column (GE Healthcare). Peak fractions of target proteins were collected and concentrated using Amicon Ultra-15 10K Centrifugal filters (Sigma-Aldrich).
Binding of TAT, Wbox2_mutant, and Wbox2 to SNX9 and β2-HAD was investigated by ITC using a MicroCal iTC₂₀₀ (GE Healthcare Life Sciences). Measurements were performed at 20°C in reaction buffer: 20 mM Hepes, 150 mM NaCl, and 1 mM TCEP, pH 7.4. Protein and peptide concentrations were determined by absorbance at 280 and 205 nm, respectively. The peptides were injected from a syringe in 21 steps up to a molar excess over the protein concentration. Titration curves were executed with NITPIC and fitted with SEDPHAT. GUSSI was further used to output the figures presented.

Chen Z., Mino R.E., Mettlen M., Michaely P., Bhave M., Reed D.K, & Schmid S.L. (2020). Wbox2: A clathrin terminal domain–derived peptide inhibitor of clathrin-mediated endocytosis. The Journal of Cell Biology, 219(9), e201908189.

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Purified Protein Complex Analysis

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Purified MxiN and Spa33 were both dialyzed into 20 mM Tris, pH 7.5, 150 mM NaCl with 10% glycerol (v/v) for SEC. The MxiN and Spa33 were combined with an excess of MxiN and the mixture was concentrated to a final volume of 2 mL for injection onto a Hi Load 26/600 Superdex 200 pg column (GE Healthcare Life Sciences). The protein absorbance at 280 nm was monitored to detect protein elution on an AKTA system (GE Healthcare Life Sciences). The eluted fractions were collected for SDS-PAGE analysis.

Tachiyama S., Skaar R., Chang Y., Carroll B.L., Muthuramalingam M., Whittier S.K., Barta M.L., Picking W.L., Liu J, & Picking W.D. (2021). Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System. Frontiers in Cellular and Infection Microbiology, 11, 682635.

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Bioconjugation of Trastuzumab to Compound 30

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Example 24

The bioconjugate according to the invention was prepared by conjugation of compound 30 as linker-conjugate to modified biomolecule 13b as biomolecule. To a solution of trastuzumab(azide)₂(13b) (15.5 mL, 255 mg, 16.47 mg/ml in PBS pH 7.4) was added DMA (1.45 mL) and compound 30 (255 μL, 40 mM solution DMA). The reaction was incubated overnight at rt followed by purification on a HiLoad 26/600 Superdex200 PG column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the reduced sample showed one major heavy chain product (observed mass 50938 Da, approximately 80% of total heavy chain fragment), corresponding to the conjugated heavy chain.

US11338043B2. Antibody-conjugates with improved therapeutic index for targeting HER2 tumours and method for improving therapeutic index of antibody-conjugates (2022-05-24). SYNAFFIX B.V. [NL]. Inventors: Sander Sebastiaan Van Berkel [NL], Jorge Merijn Mathieu Verkade [NL], Maria Antonia Wijdeven [NL], Ryan Heesbeen [NL], Petrus Josephus Jacobus Maria Van De Sande [NL], Remon Van Geel [NL], Brian Maria Gerardus Janssen [NL], Inge Catharina Josephina Hurkmans [NL], Floris Louis Van Delft [NL].

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Bioconjugate Synthesis of Trastuzumab

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Example 32

The bioconjugate according to the invention was prepared by conjugation of 76 as linker-conjugate to modified biomolecule 13d as biomolecule. To a solution of trastuzumab(6-N₃-GalNAc)₂(13d) (320 μL, 7.2 mg, 22.5 mg/ml in PBS pH 7.4) was added 76 (44.5 μL, 10 mM solution DMF) and DMF (66 μL). The reaction was incubated two times overnight at rt followed by dilution with PBS (1 mL) and concentration using an Amicon Ultra-0.5, Ultracel-10 Membrane spinfilters (Millipore). Next the conjugate was purified on a HiLoad 26/600 Superdex200 PG column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the sample after Fabricator™ treatment showed one major Fc/2 product (observed mass 25649 Da, approximately 90% of total Fc/2), corresponding to the conjugate 85.

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Trastuzumab Bioconjugation Synthesis

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Example 27

The bioconjugate according to the invention was prepared by conjugation of compound 33 as linker-conjugate to modified biomolecule 13c as biomolecule. To a solution of trastuzumab(azide)₂(13c) (14.25 mL, 250 mg, 17.55 mg/ml in PBS pH 7.4) was added compound 33 (188 μL, 40 mM solution DMA). The reaction was incubated overnight at rt followed by purification on a HiLoad 26/600 Superdex200 PG column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the reduced sample showed one major heavy chain product (observed mass 51020 Da, approximately 90% of total heavy chain fragment), corresponding to the conjugated heavy chain.

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Bioconjugation of Trastuzumab with Linker

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Example 26

The bioconjugate according to the invention was prepared by conjugation of compound 30 as linker-conjugate to modified biomolecule 13c as biomolecule. To a solution of trastuzumab(azide)₂(13c) (14.25 mL, 250 mg, 17.55 mg/ml in PBS pH 7.4) was added DMA (1.4 mL) and compound 30 (250 μL, 40 mM solution DMA). The reaction was incubated overnight at rt followed by purification on a HiLoad 26/600 Superdex200 PG column (GE Healthcare) on an AKTA Purifier-10 (GE Healthcare). Mass spectral analysis of the reduced sample showed one major heavy chain product (observed mass 50969 Da, approximately 70% of total heavy chain fragment), corresponding to the conjugated heavy chain.

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Recombinant IgG Purification Protocol

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IgG expression vectors were constructed by conjugating the heavy chains to rabbit IgG Fc by using an antibody-expressing positive control vector for IgG expression (ThermoFisher Scientific) as a template. Expi293 cells were cotransfected with expression vectors of heavy and light chains for each IgG, and the supernatant was collected 4 days after transfection. The supernatant was loaded on rProtein A Sepharose Fast Flow (GE Healthcare) equilibrated with PBS. The resin was washed with PBS, and subsequently the IgGs were eluted by 50 mM sodium citrate buffer (pH 3.0). The eluted fractions were immediately neutralized with 2M Tris-HCl (pH 8.0) and further purified by size exclusion chromatography using a HiLoad 26/600 Superdex 200-pg column (GE Healthcare) equilibrated with PBS.

Ishii M., Nakakido M., Caaveiro J.M., Kuroda D., Okumura C.J., Maruyama T., Entzminger K, & Tsumoto K. (2020). Structural basis for antigen recognition by methylated lysine–specific antibodies. The Journal of Biological Chemistry, 296, 100176.

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Gel Filtration Chromatography Optimization

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Gel filtration chromatography was performed using a HiLoad 26/600 Superdex 200 pg column (GE Healthcare, Chicago) or a Superose 6 10/300 GL column (GE Healthcare, Chicago) with TESG10N buffer. For a Superdex 200 column, 2 or 5 ml of sample was applied. Chromatography was performed at a flow rate of 1.0 ml per min for 600 min, and 5 ml fractions were collected. For a Superose 6 column, 0.2 ml of sample was applied. Chromatography was performed at a flow rate of 0.2 ml per min for 250 min, and 0.5 ml fractions were collected.

Kinugawa H., Kondo N., Komine‐Abe A., Tomita T., Nishiyama M, & Kosono S. (2020). In vitro reconstitution and characterization of pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum. MicrobiologyOpen, 9(10), e1113.

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Purification of Recombinant Gαi1 Proteins

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Purification was performed as described (8 (link)). Briefly, cells were thawed, disrupted with a microfluidizer M-110L (Microfluidics, Newton, MA), and centrifuged for 45 min at 45,000 g and 4°C to remove cell fragments. Supernatants were applied to a 25 mL nickel-nitrilotriacetic acid superflow column (Qiagen, Hilden, Germany) and eluted with buffers containing 200 mM imidazole. Fractions containing wild-type or mutant Gα_i1 were screened via SDS-PAGE, pooled, concentrated to 5 mL using a 10,000 MWCO concentrator (Amicon Ultra-15, Merck), and applied to an illustra HiLoad 26/600 Superdex 200 pg column (GE Healthcare Life Sciences, Freiburg, Germany). Peak fractions were collected, concentrated to ca. 20 mg/mL, and concentrations were determined using Bradford reagent as triplicates. Wild-type or mutant protein was aliquoted, flash-frozen in liquid nitrogen, and stored at –80°C until utilization.

Mann D., Höweler U., Kötting C, & Gerwert K. (2017). Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy. Biophysical Journal, 112(1), 66-77.

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Gel Filtration Protein Molecular Weight

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Standard molecular weight proteins from the high molecular weight Gel Filtration Calibration Kit (GE Healthcare) were dissolved with the running buffer (50 mM phosphate, 150 mM NaCl, pH 7.0) as described in the manual. Briefly, for standard proteins, 0.5 ml of each standard protein was loaded onto the HiLoad 26/600 Superdex 200 pg column (GE Healthcare) with a recommended flow rate of 2.6 ml/min in the AKTA purifier L25. Blue Dextran 2000 was used to detect the void volume. UV peaks of each protein were used to calculate the elution volume (24 (link)). Pfu Rnl and the fusion protein were loaded separately on the column and elution volumes were recorded for molecular weight comparison (25 (link)).

Yang Z., Zhang C., Lian G., Dong S., Song M., Shao H., Wang J., Zhong T., Luo Z., Jin S, & Ding C. (2022). Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase. Nucleic Acids Research, 50(13), 7560-7569.

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