Sepharose CL-2B, Sepharose CL-4B, and Sepharose CL-6B resins (Cytiva) were washed with PBS in a glass bottle. The volume of resin was washed three times with an equal volume of PBS before use. Econo-Pac Chromatography columns (Bio-Rad) were packed with resin and a frit was inserted into the column above the resin. For all columns in Figures 4 and 5 , each column was washed with 10 ml PBS (twice 5 ml at a time) prior to loading of sample. For SEC columns, resin was added until the bed volume (resin without liquid) reached 10 ml. For DMC columns, Fractogel EMD SO3- (M) (MilliporeSigma) was added as a bottom layer with 2 ml bed volume, and 10 ml of Sepharose CL-6B bed volume was added as a top layer. For TMC columns, a 2:1 by volume (of dry resin) mixture was prepared of Fractogel EMD SO3- (M) (MilliporeSigma) and Capto Core 700 (Cytiva) and 2 ml bed volume bottom layer was added to the column before 10 ml of Sepharose CL-6B bed volume was added as a top layer.
Fractogel emd so3 m
Manufactured by Merck Group
Fractogel-EMD-SO3- (M) is a sulfonic acid-functionalized separation medium used in chromatography. It is designed for the purification and separation of biomolecules, such as proteins and peptides, based on their charge characteristics. The core function of this product is to facilitate the ion-exchange separation of these target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Capto, by Cytiva (3 mentions)
Sepharose cl 4b, by GE Healthcare (3 mentions)
Miracloth, by Merck Group (2 mentions)
Emd tmae hicap m, by Merck Group (2 mentions)
Ny1102500, by Merck Group (2 mentions)
Econo pac chromatography column, by Bio-Rad (1 mentions)
Sepharose cl 6b, by Cytiva (1 mentions)
Sepharose cl 2b, by Cytiva (1 mentions)
Capto core 700, by Cytiva (1 mentions)
Captosp impres, by GE Healthcare (1 mentions)
Eshmuno s, by Merck Group (1 mentions)
Nuvia hr s, by Bio-Rad (1 mentions)
Capto adhere, by GE Healthcare (1 mentions)
Mab select sure protein a, by GE Healthcare (1 mentions)
Mabselect, by Cytiva (1 mentions)
Ny2002500, by Merck Group (1 mentions)
10 ml syringe, by BD (1 mentions)
Sp sepharose fast flow, by GE Healthcare (1 mentions)
Capto s, by GE Healthcare (1 mentions)
7 protocols using fractogel emd so3 m
1
Chromatography Column Packing Protocol
2
Purification of native FGB1 from P. indica
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To purify native FGB1 the supernatant of 7-day old P. indica cultures (2 l) grown in CM was filtrated through Miracloth (Merckmillipore) and subsequently filtrated through 0.22 μm sterile nitrocellulose filters. The obtained cell free culture filtrate was diluted 1:1 with water and supplemented with 1 mM phenylmethylsulphonyl fluoride and 15 ml 1 M sodium acetate buffer pH 5.0 per 4 l to raise the pH of the culture to pH ∼5. The culture filtrate was then applied to columns containing EMD TMAE Hicap (M) (Merck) ion exchange material. The flow through of this step was then applied to columns containing Fractogel-EMD-SO3- (M) (Merck) ion exchange material. After protein binding the column was washed with 10 volumes of 10 mM sodium acetate buffer pH 5.0. The crude FGB1 concentrate was obtained by step elution of bound proteins with 10 mM sodium acetate buffer pH 5.0 containing 1.5 M NaCl. Final purification was done by applying the SO3- eluate to a spin concentrator with a 30,000 Da cutoff. The flow through of this step was then concentrated using a 5,000 Da cutoff spin concentrator and the concentrated protein was 2 × dialysed (1 × 3hr, 1 × overnight) against 3 l of water.
3
Purification of Recombinant Antibodies
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The chemicals used in this study were obtained from J.T. Baker (Phillipsburg, NJ). Recombinant antibody fragments (Fabs), mAbs, fusion proteins, and bispecific antibodies were produced in‐house using standard CHO cell culture techniques. Recombinant murine Cathepsin L (His‐tagged) was purchased from Sino Biological, Inc. (Gaithersburg, MD). The protease inhibitors used in the study were purchased from ThermoFisher Scientific (Grand Island, NY). Chromatographic resins MabSelect SuRe™ Protein A, Capto™ SP ImpRes, and Capto™ adhere were from GE Healthcare (Piscataway, NJ); POROS™ HQ, XQ, XS, and CaptureSelect™ CH1 (IgG‐CH1, specifically designed the purification of recombinant Fab fragments and IgGs) resins were from ThermoFisher Scientific; Toyopearl® GigaCap S650 M and NH2‐750F resins were from Tosoh Bioscience (King of Prussia, PA); Eshmuno® S and Fractogel® EMD SO3[M] resins were from EMD Millipore (Burlington, MA), Nuvia™ HR‐S and C‐prime resins were from Bio‐Rad (Hercules, CA), MEP® HyperCel were from Pall (New York, NY), Natriflo® HD‐Q membrane cassette was from Natrix (Burlington, ON, Canada).
4
Purification of P. indica Proteins
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Two liters of culture filtrate of 7-day old P. indica cultures were harvested by filtration through Miracloth (Merckmillipore) and subsequent filtration through 0.22 μm sterile nitrocellulose filters. The obtained cell free culture filtrate was pH adjusted with phosphate buffer to pH 7.5 and separated via 40 ml Fractogel-EMD-SO3- (M) (Merck) and EMD TMAE Hicap (M) (Merck) ion exchange material into acidic- and alkaline-protein fractions. The obtained protein fractions were dialysed against 10 mM sodium acetate buffer containing 150 mM NaCl pH 5.0 and 400 μl were incubated with 50 μl sepharose beads for 30 min at room temperature before washing with an excess of binding buffer. Subsequently, the sepharose beads were boiled in Laemmli SDS-buffer and bound proteins were analysed by SDS–polyacrylamide gel electrophoresis (PAGE).
5
Extracellular Vesicle Isolation from Plasma
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SEC and DMC columns were prepared as described previously (Van Deun et al., 2020 ). Sepharose CL‐4B (GE Healthcare) and Fractogel EMD SO3− (M) (Merck Millipore) resins were washed three times with PBS buffer. A nylon net with 20 μm pore size (NY2002500, Merck Millipore) was placed on the bottom of a 10 mL syringe (BD307736, BD Biosciences). For the SEC column, this was followed by stacking of 10 mL washed Sepharose. For the DMC column, 2 mL of Fractogel was stacked first, followed by careful layering of 10 mL Sepharose on top. After adding 0.5 mL plasma sample, the column was eluted by constant addition of fresh PBS. After discarding the void volume, which was 3 mL for SEC and 3.5 mL for DMC, 2 mL of EV‐containing eluate was collected and concentrated to 100 μL using Amicon Ultra‐2 10K filters (Merck Millipore).
6
Extracellular Vesicle Isolation Using Size Exclusion and Ion Exchange Chromatography
7
Size Exclusion Chromatography Column Preparation
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Sepharose CL-4B (GE Healthcare) and Fractogel EMD SO3- (M) (Millipore Sigma) resins were used after double washing with PBS. A nylon net filter with 11 μm pore size (NY1102500, Millipore Sigma) was cut and placed on the bottom of a 10 mL syringe (BD Biosciences). For the SE-only column, 10 mL of washed Sepharose matrix was stacked on the syringe. In case of the dual-mode column, 2 mL of washed Fractogel resin was loaded on the syringe first, and then 10 mL of washed Sepharose gel was stacked on top. Prepared columns were allowed to settle at least for 24 hours and stored at 4 °C until use.
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