Sf9 cells were used to generate baculovirus stocks using the Bac-to-Bac system (Life Technologies), according to the manufacturer's instructions. A baculovirus stock carrying the gene for DNMT1 was used to infect 4–12 L of Sf9 cells at a density of 2 million cells/mL in Sf-900 III SFM (Invitrogen cat. # 12658–027). Multiple 1 L cell cultures in 4 L flasks (VWR cat. # 32645–044) were incubated for 72 h at 27°C, 130 revolutions per minute (rpm). Cells were harvested by centrifugation for 20 min at 2000 relative centrifugal force (RCF) (JLA-8.1, Beckman) at 4°C, then frozen in liquid nitrogen and stored at −80°C until protein purification.
Jla 8
Manufactured by Beckman Coulter
Sourced in France
The JLA 8.1000 is a high-performance floor-standing ultracentrifuge designed for a wide range of laboratory applications. It features a maximum speed of 100,000 rpm and a maximum RCF of 803,500 x g, enabling efficient separation and purification of various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Sf 900 3 sfm, by Thermo Fisher Scientific (2 mentions)
Bac to bac system, by Thermo Fisher Scientific (2 mentions)
Avanti j 2 xpi centrifuge, by Beckman Coulter (2 mentions)
Innova 44, by Eppendorf (1 mentions)
Completetm mini protease inhibitor cocktail, by Roche (1 mentions)
Ja 25, by Beckman Coulter (1 mentions)
Pierce bicinchoninic acid bca protein assay, by Thermo Fisher Scientific (1 mentions)
Minisart filter, by Sartorius (1 mentions)
11 protocols using jla 8
1
Baculovirus Production for DNMT1 Purification
2
Production of Recombinant Human PRC2 Complex
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Expression of human PRC2 was carried out as previously described (23 (link)), with some modifications. Sf9 cells where used to generate baculovirus stocks using the Bac-to-Bac system (Life Technologies), according to the manufacturer's instructions. Appropriate ratios of baculovirus stocks carrying genes for the four (with the exclusion of AEBP2) or five PRC2 subunits were used to co-infect 4–12 l of Sf9 cells at a density of 2 million cells/ml in Sf-900™ III SFM (Invitrogen cat # 12658–027). Multiple 1 l cell cultures in 4 l flasks (VWR cat # 32645–044) were incubated 72 h at 27°C, 130 revolutions per minute (rpm). Cells were harvested by centrifugation for 20 min at 2000 relative centrifugal force (RCF) (JLA-8.1, Beckman) at 4°C, frozen in liquid nitrogen and stored at −80°C until protein purification.
3
Purification of Sortase A Variants
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Chemically competent E. coli cells were transformed with a pET28 vector containing the genes coding for His6-tagged Sortase
A and variants8M and S11 . Cells were grown
in TB medium at 37 °C, and protein expression was induced with
0.5 mM IPTG and performed at 25 °C overnight. Cells were harvested
using centrifugation (Beckman Coulter JLA8.1, 4000 rpm, 20 min, 4
°C) and resuspended in lysis buffer (20 mM HEPES, pH 7.5, 150
mM NaCl, 5 mM CaCl2, 0.5 mM TCEP). Cell lysis was performed
using a microfluidizer (Microfluidics LM10) at 15000 psi. The resulting
cell lysate was cleared of debris by centrifugation (Beckman Coulter
JA25.50, 21000 rpm, 45 min, 4 °C). Cleared cell lysate was loaded
on a pre-equilibrated Ni-affinity column (Cytiva, HisTrap FF crude
5 mL). The column was washed with eight column volumes (CV) of wash
buffer (50 mM HEPES, pH 7.5, 400 mM NaCl, 5% glycerol, 20 mM imidazole,
0.5 mM DTT) and protein eluted by circulating PreScission protease
overnight for on-column digest. Eluted protein was subjected to size
exclusion chromatography (SEC) (GE Healthcare, Äkta Pure, HiLoad
16/600 S75) using SEC buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5
mM TCEP). Protein containing fractions were pooled, concentrated (3
kDa MWCO amicon centrifugal filter, Merck), and flash frozen in liquid
nitrogen. Sortase A wild type was purified as previously reported.19 (link)
A and variants
in TB medium at 37 °C, and protein expression was induced with
0.5 mM IPTG and performed at 25 °C overnight. Cells were harvested
using centrifugation (Beckman Coulter JLA8.1, 4000 rpm, 20 min, 4
°C) and resuspended in lysis buffer (20 mM HEPES, pH 7.5, 150
mM NaCl, 5 mM CaCl2, 0.5 mM TCEP). Cell lysis was performed
using a microfluidizer (Microfluidics LM10) at 15000 psi. The resulting
cell lysate was cleared of debris by centrifugation (Beckman Coulter
JA25.50, 21000 rpm, 45 min, 4 °C). Cleared cell lysate was loaded
on a pre-equilibrated Ni-affinity column (Cytiva, HisTrap FF crude
5 mL). The column was washed with eight column volumes (CV) of wash
buffer (50 mM HEPES, pH 7.5, 400 mM NaCl, 5% glycerol, 20 mM imidazole,
0.5 mM DTT) and protein eluted by circulating PreScission protease
overnight for on-column digest. Eluted protein was subjected to size
exclusion chromatography (SEC) (GE Healthcare, Äkta Pure, HiLoad
16/600 S75) using SEC buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5
mM TCEP). Protein containing fractions were pooled, concentrated (3
kDa MWCO amicon centrifugal filter, Merck), and flash frozen in liquid
nitrogen. Sortase A wild type was purified as previously reported.19 (link)
4
Expression and Purification of Isotopically Labeled α-Synuclein
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Escherichia coli strain BL21(DE3) plysS was transformed with pET24–α-syn vector by electroporation and plated onto Luria broth agar plate containing kanamycin (30 μg/ml). A preculture in 5 ml of Luria broth medium was inoculated with one clone and incubated at 37°C under 200 rpm shaking for 4 hours. The expression on α-syn was carried out in M9 minimal medium containing 13C glucose (2 g/liter) and 15NH4Cl (1 g/liter) as carbon and nitrogen sources. Cells from the Luria broth preculture were recovered by centrifugation (1000g, 10 min) and used for inoculating 200 ml of M9 medium. Cells were grown overnight at 37°C under 200 rpm shaking and then diluted in 2 liters of culture. Protein expression was induced by adding 1 mM isopropyl-β-d -thiogalactopyranoside during exponential phase, evaluated at an optical density at 600 nm reaching 0.8. Cells were harvested after 4 to 5 hours of culture at 37°C by 6000g centrifugation (JLA 8.1, Beckman Coulter), and pellet was kept at −20°C before purification. The site-specific nonphosphorylatable mutant S129A was obtained by site-directed mutagenesis of pET24–α-syn.
5
Overexpression of FadD13 Variants in E. coli
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The plasmid vector with
an insert (FadD13/hydrophobic
variant/negative variant) was transformed into E. coli expression strains BL21(DE3) or T7 Express LysY. Terrific Broth
supplemented with 50 μg/mL antibiotic (carbenicillin or tetracycline),
0.4% glycerol, and 0.01% Antifoam 204 was inoculated with a starter
culture at a 1:100 dilution. The cultures were grown in a LEX bubbling
system at 37 °C to a density OD600 of 2.0–2.5.
Overexpression was induced by addition of 0.25 mM IPTG. The temperature
was decreased to 20 °C, and the cultures were grown for an additional
∼18–20 h. The cells were harvested via centrifugation
at 7500g for 15 min (JLA 8.1000, Beckman Coulter).
The resulting cell pellet was transferred to 50 mL falcon tubes, frozen,
and stored at −20 °C.
an insert (FadD13/hydrophobic
variant/negative variant) was transformed into E. coli expression strains BL21(DE3) or T7 Express LysY. Terrific Broth
supplemented with 50 μg/mL antibiotic (carbenicillin or tetracycline),
0.4% glycerol, and 0.01% Antifoam 204 was inoculated with a starter
culture at a 1:100 dilution. The cultures were grown in a LEX bubbling
system at 37 °C to a density OD600 of 2.0–2.5.
Overexpression was induced by addition of 0.25 mM IPTG. The temperature
was decreased to 20 °C, and the cultures were grown for an additional
∼18–20 h. The cells were harvested via centrifugation
at 7500g for 15 min (JLA 8.1000, Beckman Coulter).
The resulting cell pellet was transferred to 50 mL falcon tubes, frozen,
and stored at −20 °C.
6
Bacterial Protein Expression and Purification
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For protein expression, 2 l LB were inoculated with 20 ml of an overnight culture containing 100 μg/ml ampicillin and incubated at 37 °C until an absorbance reached a value of 0.6 to 0.8 at 600 nm. Overexpression was induced by adding IPTG to a final concentration of 1 mM and further incubation for 4 h at 37 °C. Cells were harvested by centrifugation (Beckman Coulter; JLA 8.1000; 4000g, 20 min, 4 °C), and pellets were resuspended and washed with 30 ml buffer (50 mM Tris [pH 8], 300 mM NaCl, and 20 mM Imidazole). After recentrifugation (Eppendorf 5920R; S-4x750; 4000g, 1 h, 4 °C), the washed pellets were stored at −20 °C until further use.
7
Recombinant Protein Production of 13C/15N α-Synuclein
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Escherichia coli strain BL21(DE3) plysS was transformed with pET24–α-Syn vector by electroporation and plated onto a Luria broth agar plate containing kanamycin (30 μg/mL). A preculture in 5 mL of Luria broth medium was inoculated with one clone and incubated at 37 °C under 200 rpm shaking for 4 h. The expression on α-Syn was carried out in M9 minimal medium containing 13C glucose (2 g/L) and 15NH4Cl (1 g/L) as carbon and nitrogen sources. Cells from the Luria broth preculture were recovered by centrifugation (1000× g, 10 min) and used for inoculating 200 mL of M9 medium. Cells were grown overnight at 37 °C under 200 rpm shaking and then diluted in 2 L of culture. Protein expression was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside during the exponential phase, evaluated at an optical density at 600 nm reaching 0.8. Cells were harvested after 4 to 5 h of culture at 37 °C by 6000× g centrifugation (JLA 8.1000, Beckman Coulter, Villepinte, France), and pellet was kept at −20 °C before purification.
8
Overexpression of Recombinant Proteins in E. coli
9
Overexpression and Isotopic Labeling of α-Synuclein
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Escherichia coli strain BL21(DE3) plysS was transformed with pET24–α-Syn vector by electroporation and plated onto a Luria broth agar plate containing kanamycin (30 μg/mL). A preculture in 5 mL of Luria broth medium was inoculated with one clone and incubated at 37 °C under 200 rpm shaking for 4 h. The expression on α-Syn was carried out in M9 minimal medium containing 13C glucose (2 g/L) and 15NH4Cl (1 g/L) as carbon and nitrogen sources. Cells from the Luria broth preculture were recovered by centrifugation (1000× g, 10 min) and used for inoculating 200 mL of M9 medium. Cells were grown overnight at 37 °C under 200 rpm shaking and then diluted in 2 L of culture. Protein expression was induced by adding 1 mM isopropyl-β-d -thiogalactopyranoside during the exponential phase, evaluated at an optical density at 600 nm reaching 0.8. Cells were harvested after 4 to 5 h of culture at 37 °C by 6000× g centrifugation (JLA 8.1000, Beckman Coulter, Villepinte, France), and pellet was kept at −20 °C before purification.
10
Bacterial Cell Lysis and Protein Quantification
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B. megaterium and B. subtilis cultures (1 L/cell type) were grown in nutrient broth 3 (NB3) and Luria Broth (Miller) media, respectively, at 37 °C until OD600 reached mid-exponential. The cells were then harvested via centrifugation for 20 min at 6200× g at 4 °C in a Beckman JLA 8.1000 rotor. The pellet was resuspended in 50 mM Tris pH 8.0, 25 mM NaCl, and 1x PIC (cOmpleteTM Mini Protease Inhibitor Cocktail—Roche, Welwyn Garden City, UK). To lyse the cells, sonication was performed on ice by using Soniprep150 (15 cycles—15 s pulse ON/20 s pulse OFF). Following sonication, the samples were centrifuged for 30 min at 39,000× g at 4 °C in a Beckman JA25.50 rotor. The cell lysates were then filtered by using a 0.2 μm Minisart® filter (Sartorius, Epson, UK). The proteins within the lysate were quantified with the Pierce™ Bicinchoninic acid (BCA) Protein Assay (Thermo Scientific) by using the CLARIOstar Plus spectrophotometer (CLARIOstar® software version 5.61).
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