Recombinant α-synuclein protein was prepared as described previously.64 (link) Briefly, full-length human wild-type α-synuclein (Y136 codon-modified) encoded in a plasmid pRK17262 (link) was expressed in E. coli BL21(DE3) and purified by boiling, Q sepharose ion exchange chromatography (Q Sepharose Fast Flow, Cytiva), ammonium sulfate precipitation and dialysis against 30 mM Tris-HCl (pH7.5). The high degree of purification (>90%) was confirmed by SDS-PAGE and CBB staining.
Q sepharose fast flow
Manufactured by Cytiva
Sourced in United States
Q Sepharose Fast Flow is a strong anion exchange chromatography resin designed for the purification of biomolecules. It consists of highly cross-linked agarose beads with quaternary ammonium functional groups, providing a high dynamic binding capacity and fast flow properties.
Automatically generated - may contain errors
Lab products found in correlation
Capto, by Cytiva (2 mentions)
Kta avant, by Cytiva (1 mentions)
Kta pure system, by Cytiva (1 mentions)
Membrane filter, by Sartorius (1 mentions)
0.45 μm filter units, by Sartorius (1 mentions)
Capto core 700, by Cytiva (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
5 protocols using q sepharose fast flow
1
Purification of Recombinant α-Synuclein
2
Purification of His-tagged Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The bacterial culture was centrifuged at 5000× g for 30 min and the supernatant was discarded. The pellet was resuspended in lysis buffer (20 mM phosphate buffer pH 7.4, 500 mM NaCl, 1 mM PMSF, 0.1% Triton X-100). Bacterial cells were disrupted by high-pressure homogenizer (PANDA Plus 2000, GEA, Düsseldorf, Germany) through three passages at 1200 bar, keeping the cell suspension in an ice bath during the passages. All purification methods were conducted using 15 to 20 g of bacterial biomass.
IMAC: The chromatography column (XK26) was packed with 50 mL IMAC-Sepharose 6 Fast Flow resin (Cytiva, Marlborough, MA, USA), the resin was charged with Ni2+ and chromatography was performed on ÄKTA Avant (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with a buffer containing 20 mM phosphate, pH 7.4, 500 mM NaCl buffer and 10 mM imidazole. Elution was performed with increasing concentrations of imidazole (50 mM, 150 mM, and 500 mM).
Q-Sepharose: The chromatography column (XK26) was packed with 74 mL of Q-Sepharose Fast Flow (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with 50 mM Tris-HCl, pH 8.0. Elution was performed with increasing concentrations of NaCl (0 to 0.7 M).
IMAC: The chromatography column (XK26) was packed with 50 mL IMAC-Sepharose 6 Fast Flow resin (Cytiva, Marlborough, MA, USA), the resin was charged with Ni2+ and chromatography was performed on ÄKTA Avant (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with a buffer containing 20 mM phosphate, pH 7.4, 500 mM NaCl buffer and 10 mM imidazole. Elution was performed with increasing concentrations of imidazole (50 mM, 150 mM, and 500 mM).
Q-Sepharose: The chromatography column (XK26) was packed with 74 mL of Q-Sepharose Fast Flow (Cytiva, Marlborough, MA, USA). The column was previously equilibrated with 50 mM Tris-HCl, pH 8.0. Elution was performed with increasing concentrations of NaCl (0 to 0.7 M).
3
Immunotoxin Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids for the heavy and the light chain of the wild-type (wt) immunotoxin were kindly provided by Dr Ira Pastan. For the PE mutant variants, codon-optimized gene blocks were ordered (IDT, Coralville, IA, USA) and cloned into the wt expression plasmid. Expression and purification of the immunotoxin were performed as described previously [49 (link)]. Briefly, immunotoxin heavy and light chains were expressed individually in BL21 E. coli (NEB, Ipswich, MA, USA), isolated as inclusion bodies, refolded for 32 h at pH 10.0 (11), dialyzed for two to three days, and then purified with a three-step chromatography protocol including two distinct ion exchange columns (Q Sepharose Fast Flow, Cytiva and Capto HiRes Q, Cytiva) followed by size exclusion column (Superdex 75 Increase GL, Cytiva) using an ÄKTApure system (Cytiva).
4
Purification of Yellow Fever Vaccine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The processes of YFV production and purification are similar to those described by Pato and coworkers [10 (link)]. The working virus seed of YF 17D-213 infects the Vero cells (WHO-approved cell line) and undergoes bioreactor culturing. The supernatant of the culture medium containing the virus was treated with β-propriolactone for 24 h at room temperature to inactivate virus, and then concentrated 100 times by using a molecular weight cutoff of 300 kDa membrane filter (Sartorius, Germany), followed by downstream chromatography purification. Chromatography was performed using Akta pilot, operated with the UNICORN software (https://www.cytivalifesciences.com/en/us/shop/chromatography/software/unicorn-7-p-05649 ) (Cytiva, Marlborough, MA, USA). Ion exchange (Q Sepharose Fast Flow) and Capto Core700 (Cytiva, USA) resin prepacked in an XK50 Column were used for capturing and polishing, respectively, and the entire purification process. The purified sample was subsequently filtered using 0.45 μm filter units (Sartorius, Goettingen, Germany).
5
pH-Dependent Plasma Fractionation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
0.5 mL
of platelet-deprived plasma was first buffer exchanged with 10 mM
buffers of respective pHs by using a 7 kDa molecular weight cutoff
(MWCO) Zeba Spin Desalting Column (Thermo Scientific). A buffer pH
of 5.4 was constituted by an acetate buffer, while phosphate buffers
were used to prepare the buffers of pH 6.4 and pH 7.4. Buffers of
pH 8.4 and 9.4 were formulated with tris and carbonate/bicarbonate
buffers, respectively. All buffer concentrations were adjusted to
10 mM in this study. Simultaneously, 0.5 mL of positively charged
anion exchanger, Q Sepharose Fast Flow (Cytiva) was equilibrated with
buffers of various pHs. Afterward, 0.5 mL of buffer exchanged plasma
was thoroughly mixed with 0.5 mL of buffer-equilibrated positively
charged beads left at room temperature for 10 min. The supernatant,
which contains unbound plasma components, was then collected, and
its osmolarity was made equivalent to 1× PBS by using 10×
PBS.
For analysis of plasma constituents that have been bound
to positively charged beads, the supernatant which contains the unbound
plasma constituents was first removed, and the positively charged
beads were washed with DI water 3 times. Afterward, a 1 M NaCl solution
was added to the washed beads to detach the entire bound plasma constituents
from the positively charged beads.
of platelet-deprived plasma was first buffer exchanged with 10 mM
buffers of respective pHs by using a 7 kDa molecular weight cutoff
(MWCO) Zeba Spin Desalting Column (Thermo Scientific). A buffer pH
of 5.4 was constituted by an acetate buffer, while phosphate buffers
were used to prepare the buffers of pH 6.4 and pH 7.4. Buffers of
pH 8.4 and 9.4 were formulated with tris and carbonate/bicarbonate
buffers, respectively. All buffer concentrations were adjusted to
10 mM in this study. Simultaneously, 0.5 mL of positively charged
anion exchanger, Q Sepharose Fast Flow (Cytiva) was equilibrated with
buffers of various pHs. Afterward, 0.5 mL of buffer exchanged plasma
was thoroughly mixed with 0.5 mL of buffer-equilibrated positively
charged beads left at room temperature for 10 min. The supernatant,
which contains unbound plasma components, was then collected, and
its osmolarity was made equivalent to 1× PBS by using 10×
PBS.
For analysis of plasma constituents that have been bound
to positively charged beads, the supernatant which contains the unbound
plasma constituents was first removed, and the positively charged
beads were washed with DI water 3 times. Afterward, a 1 M NaCl solution
was added to the washed beads to detach the entire bound plasma constituents
from the positively charged beads.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!