To purify PPV VLPs by IEX chromatography, yeast cells were suspended in PBS buffer pH7.4 and disrupted by high-pressure homogenization. Cell lysates were subsequently adjusted to pH 4.0. After centrifugation at 10,000 rpm, 4 °C for 30 min, the supernatants of pH adjusted cell lysate were loaded onto an XK 50/30 column (GE Healthcare) packed with 400 mL of Capto S ImpAct resin. Binding VLPs were eluted with 20 mM sodium acetate buffer containing 500 mM NaCl. To elevate the recovery of VLPs, the precipitates of pH adjusted cell lysates were redissolved in an equal volume of 20 mM Tris-HCl buffer pH 8.0. Through centrifugation, the clarified supernatant were loaded onto an XK 50/30 column packed with 400 mL of Capto Q XP resins. After elution with 20 mM Tris-HCl buffer pH 8.0 plus 500 mM NaCl, fractions were diafiltrated for 10 volumes of PBS on ÄKTA flux (GE Healthcare, USA) equipped with a 750 kDa column (11-0005-50, GE healthcare). Further polishing purification of PPV VLPs was performed on an AKTA Purifier 100 (GE Healthcare, USA) using a HiPrep™ 26/60 Sephacryl® S-500 HR column (GE Healthcare). About 4 ml IEX purified sample was injected and eluted with PBS at a rate of 0.5 mL/min. Protein concentration was measured by the BCA Protein Assay Kit (23,250, Thermo Fisher Scientific).
Xk 50 30 column
Manufactured by GE Healthcare
Sourced in United States
The XK 50/30 column is a liquid chromatography column designed for laboratory use. It is a versatile instrument that can be used for a variety of separation techniques, including size exclusion chromatography, ion exchange chromatography, and affinity chromatography. The column has a diameter of 50 mm and a bed height of 30 cm, providing a large separation capacity. The column is constructed of durable materials and is compatible with a wide range of solvents and buffers.
Automatically generated - may contain errors
Lab products found in correlation
Hiprep 26 60 sephacryl s 500 hr column, by GE Healthcare (2 mentions)
Hiprep, by Cytiva (2 mentions)
Capto, by Cytiva (2 mentions)
Akta purifier 100, by GE Healthcare (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Kta flux, by GE Healthcare (1 mentions)
P4.1s pump, by Knauer (1 mentions)
Akta puri er 100, by GE Healthcare (1 mentions)
3 protocols using xk 50 30 column
1
Purification of PPV VLPs by IEX and SEC
2
Packed-bed Biocatalytic Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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This was carried out continuously in a packed-bed
reactor scaled up to ∼400 mL working volume. A XK50/30 column
(ID 50 mm, GE Healthcare, Chicago, Illinois, USA) was filled with
PAM-I particles in the size range of 0.25–2.00 mm. The bed
height was ∼20.3 cm. The column was connected to a circulating
water bath for temperature control at 40 °C.16 (link) Feed was from an Azura P4.1S pump (Knauer, Berlin, Germany).
The packed bed was washed with water and substrate solution (0.35
M sucrose, 1.9 M glycerol) at 1 mL min–1. Synthesis
was done with a substrate delivered at 3 mL min–1 (space velocity of 0.45 h–1) for ∼16–17
h.
reactor scaled up to ∼400 mL working volume. A XK50/30 column
(ID 50 mm, GE Healthcare, Chicago, Illinois, USA) was filled with
PAM-I particles in the size range of 0.25–2.00 mm. The bed
height was ∼20.3 cm. The column was connected to a circulating
water bath for temperature control at 40 °C.16 (link) Feed was from an Azura P4.1S pump (Knauer, Berlin, Germany).
The packed bed was washed with water and substrate solution (0.35
M sucrose, 1.9 M glycerol) at 1 mL min–1. Synthesis
was done with a substrate delivered at 3 mL min–1 (space velocity of 0.45 h–1) for ∼16–17
h.
3
Purification of PPV VLPs by IEX Chromatography
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To purify PPV VLPs by IEX chromatography, yeast cells were suspended in PBS buffer pH7.4 and disrupted by high-pressure homogenization. Cell lysates were subsequently adjusted to pH 4.0. After centrifugation at 10,000 rpm, 4 °C for 30 min, the supernatants of pH adjusted cell lysate were loaded onto an XK 50/30 column (GE Healthcare) packed with 400 ml of Capto S ImpAct resin. Binding VLPs were eluted with 20mM sodium acetate buffer containing 500 mM NaCl. To elevate the recovery of VLPs, the precipitates of pH adjusted cell lysates were redissolved in an equal volume of 20mM Tris-HCl buffer pH 8.0. Through centrifugation, the clari ed supernatant were loaded onto an XK 50/30 column packed with 400 ml of Capto Q XP resins. After elution with 20mM Tris-HCl buffer pH 8.0 plus 500 mM NaCl, fractions were dia ltrated for 10 volumes of PBS on ÄKTA ux (GE Healthcare, USA) equipped with a 750 kDa column (11-0005-50, GE healthcare). Further polishing puri cation of PPV VLPs was performed on an AKTA Puri er 100 (GE Healthcare, USA) using a HiPrep™ 26/60 Sephacryl® S-500 HR column (GE Healthcare). About 4ml IEX puri ed sample was injected and eluted with PBS at a rate of 0.5ml/min. Protein concentration was measured by the BCA Protein Assay Kit (23250, Thermo Fisher Scienti c).
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