Enterococcus faecalis ATCC V583 strain was purchased from Cryosite (NSW, Australia) and maintained on Columbia blood agar (Oxoid, Melbourne, Australia) at 37 °C. Culture purity was periodically checked by culturing onto bile aesculin agar (Oxoid). About 1000 mL of sterile Todd Hewitt broth (THB) (Oxoid), was inoculated with 1 mL of an overnight broth and incubated at 37 °C for 3 days. Bacteria were harvested by centrifugation (6000
Bacterial protease inhibitor cocktail
Manufactured by Merck Group
Sourced in United States
The Bacterial Protease Inhibitor Cocktail is a laboratory product designed to inhibit proteases often found in bacterial samples. This mixture of compounds helps preserve the integrity of proteins during extraction and analysis procedures.
Automatically generated - may contain errors
Lab products found in correlation
Bile aesculin agar, by Thermo Fisher Scientific (1 mentions)
Columbia blood agar, by Thermo Fisher Scientific (1 mentions)
Ni nta bead, by Qiagen (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Image lab software package, by Bio-Rad (1 mentions)
Gelcode blue safe stain, by Thermo Fisher Scientific (1 mentions)
Emulsiflex, by Avestin (1 mentions)
Casein peptone, by Merck Group (1 mentions)
Soya peptone, by Merck Group (1 mentions)
2 2 bipyridyl, by Merck Group (1 mentions)
Dipotassium hydrogen phosphate, by Merck Group (1 mentions)
Fecl3, by Merck Group (1 mentions)
Lysozyme, by Roche (1 mentions)
Isopropyl β d thiogalactoside iptg, by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Sodium chloride (nacl), by Sangon (1 mentions)
Tryptone, by Sangon (1 mentions)
Yeast extract, by Sangon (1 mentions)
7 protocols using bacterial protease inhibitor cocktail
1
Enterococcus faecalis Lysis and Protein Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
Enterococcus faecalis ATCC V583 strain was purchased from Cryosite (NSW, Australia) and maintained on Columbia blood agar (Oxoid, Melbourne, Australia) at 37 °C. Culture purity was periodically checked by culturing onto bile aesculin agar (Oxoid). About 1000 mL of sterile Todd Hewitt broth (THB) (Oxoid), was inoculated with 1 mL of an overnight broth and incubated at 37 °C for 3 days. Bacteria were harvested by centrifugation (6000g ), at 4 °C for 20 min. Cells were washed twice with saline (0.9% w/v) at 4 °C and cells were finally resuspended in 12 mL of ice cold saline. Cells were lysed by two passes (60 000 kPa) through a SLM Aminco French Press (Thermo Fisher, Waltham, MA, USA). Endogenous proteinase activity was controlled during lysis by the addition of 100 μL of bacterial protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Nucleic acids were then degraded by the addition of deoxyribonuclease I (2000 Units), ribonuclease A (1000 Units), and MgCl2 (50 mm ), and incubated on ice for 60 min. Intact cells were removed by centrifuging twice (8000 g at 4 °C for 5 min) and removing the supernatant.
+ Expand
Enterococcus faecalis ATCC V583 strain was purchased from Cryosite (NSW, Australia) and maintained on Columbia blood agar (Oxoid, Melbourne, Australia) at 37 °C. Culture purity was periodically checked by culturing onto bile aesculin agar (Oxoid). About 1000 mL of sterile Todd Hewitt broth (THB) (Oxoid), was inoculated with 1 mL of an overnight broth and incubated at 37 °C for 3 days. Bacteria were harvested by centrifugation (6000
2
Extraction and Purification of Photorhabdus Toxin Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial cell pellets were obtained from a 2-liter culture of the two Photorhabdus types HRM1 and HS1 fermentations, separately for 48h according to Sheets et al. (2011) with some modifications. The pellets were suspended in 50mM Tris-HCl (pH 8.0), 100mM NaCl, 1mM DTT, 10% glycerol, lysozyme (0.6mg/ml) and bacterial protease inhibitor cocktail (Sigma, St. Louis). A small amount of glass beads (0.5mm diameter), were added and then bacterial cells were disrupted by sonication then centrifuged at 10,000g for 60min at 4 °C. Supernatants, including toxin complexes (TCs), were then collected into Eppendorf tubes and subjected to protein concentration measurement using Coomassie Blue Protein Assay Reagent (ICI Americas, Inc.) according to the manufacturer’s instructions, then stored at −20 °C. The total protein was estimated by the method described by Bradford (1976) (link), and bovine serum albumin was used for the calibration curve. TCs were then stored in liquid nitrogen until used for larvicidal bio-assay screening.
3
Purification of His-tagged Menin Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli BL21 transformed with His-menin (WT, S122A, S487A, or S487D) was incubated at 25°C and then induced by 0.25 mM IPTG for 4 h. Cells were harvested and lysed with 1% Triton X-100 in PBS, 1.0 mM PMSF, 4 µg/ml bacterial protease inhibitor cocktail (Sigma), and 100 µg/ml lysozyme. Lysates were spun down, and the supernatant was added to Ni-NTA beads (Qiagen) overnight at 4°C. After washing with 1 mM imidazole in PBS, proteins were eluted from Ni-NTA beads with 150 mM imidazole in PBS.
4
Purification and Quantification of Bacterial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 5 mL starter E. coli culture was grown for 8 h at 37 °C, from which the equivalent of 1 mL of OD600 = 0.1 was used to inoculate an overnight 40 mL culture, induced with 50 μM IPTG and grown at 37 °C. The equivalent to 40 mL of OD600 = 0.6 was collected by centrifugation (6000×g for 10 min), taken up in 5 mL TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) buffer with bacterial protease inhibitor cocktail (Sigma, USA) and lysed with three passes of the Emulsiflex (Avestin, USA) set at 60psi. Lysates were clarified by centrifugation (10,000×g for 10 min) and proteins from the resultant supernatant were separated at 150 V for 150 min in non-denaturing buffer (25 mM Tris, 50 mM glycine). Proteins were fixed with protein fixing solution (50% methanol, 10% acetic acid, 40% H2O; v/v/v) for no longer than 30 min, washed with H2O, incubated with GelCode Blue Safe Stain (ThermoFisher, USA), washed with H2O and visualised with the BioRad ChemiDoc XRS + system (BioRad, USA). Bands were quantified using the Image Lab software package (Bio-Rad, ver 6.1.0 build 7).
5
Bacterial Growth Under Iron Depletion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial strains were grown on tryptic soy agar for 48 h at 22 °C. A single colony of each strain was inoculated into 10 mL of tryptic soy broth [Casein peptone (17 g), dipotassium hydrogen phosphate (2.5 g), glucose (2.5 g), sodium chloride (5 g), soya peptone (3 g) per liter, Sigma, Germany] and incubated for 17 h at 22 °C with shaking (150 rpm). These starter cultures were then diluted with fresh sterile tryptic soy broth to an optical density (OD 600) of 0.10 ± 0.05. Five hundred microlitres of the diluted starter cultures were inoculated in duplicates, into 25 mL of tryptic soy broth with or without iron chelator, 2,2′-bipyridyl, MW 156.18 g/mol (Sigma, Germany). Iron depletion was attained by the addition of 100 μM of 2,2′-bipyridyl to the broth. Cultures were grown overnight at 22 °C with shaking (150 rpm) until the late log phase. Cells were harvested by centrifugation at 4000 rpm for 10 min at 4 °C, then washed three times with phosphate buffered saline containing bacterial protease inhibitor cocktail (Sigma, Germany) and stored at −80 °C.
6
Production and Iron Loading of Recombinant H-Ferritin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant H-ferritin was prepared as previously described [32 (link)]. Briefly, wild-type human H-ferritin containing a poly-His tag was subcloned into the pET30a(+) vector, to be produced in BL21 Escherichia coli. Isopropyl-β-D-thio-galactoside (IPTG, Sigma) was used to induce expression. Following induction, bacteria were lysed in a mixture of Bugbuster (Novagen), benzonase nuclease (VWR), bacterial protease inhibitor cocktail (Sigma) and lysozyme (Roche). H-ferritin protein was purified using a nickel column (GE Healthcare Bio-Sciences) according to the manufacturer’s instructions. Identity of H-ferritin was verified by western blot (data not shown).
Apo-transferrin (apo-Tf, Sigma) and H-ferritin were iron loaded with ferric chloride hexahydrate (FeCl3, Sigma), nitrilotriacetic acid (NTA, Sigma), and sodium bicarbonate complexed at a ratio of 100 μL NTA: 13.4 μL FeCl3: 23.3 μL NaHCO3. This solution was allowed to complex for 30 minutes to create the Fe-NTA complex. Apo-Tf and H-ferritin were incubated with the Fe-NTA complex for 30 additional minutes to allow for sufficient iron loading [22 (link),23 (link)].
Apo-transferrin (apo-Tf, Sigma) and H-ferritin were iron loaded with ferric chloride hexahydrate (FeCl3, Sigma), nitrilotriacetic acid (NTA, Sigma), and sodium bicarbonate complexed at a ratio of 100 μL NTA: 13.4 μL FeCl3: 23.3 μL NaHCO3. This solution was allowed to complex for 30 minutes to create the Fe-NTA complex. Apo-Tf and H-ferritin were incubated with the Fe-NTA complex for 30 additional minutes to allow for sufficient iron loading [22 (link),23 (link)].
7
Recombinant Protein Purification from E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pET-21b based pTEV8 constructs of His-tagged CLOCKDN, ASS1, IMPDH2, ALDH1A1 were introduced into BL21 (DE3) E.coli cells. The transformants were grown at 37 C in LB medium (10 g/L tryptone, 5g/L yeast extract, 10 g/L NaCl, all from Sangon Biotech, Shanghai) untill the OD600 of medium reached 0.6. Protein expression in E.coli was stimulated by IPTG (Isopropyl b-D-1-thiogalactopyranoside, Sangon Biotech, Shanghai) for 16h at 16 C. E.coli cells were spinned down and re-suspended in TBS buffer (50mM Tris-HCl pH 7.5, 150mM NaCl) containing 5 mg/ml DNase I (New England Biolabs) and bacterial protease inhibitor cocktail (Sigma-Aldrich), then lysed in a high-pressure homogenizer (JN-3000 PLUS low temperature ultra-high pressure continuous flow cell disrupter, JNBIO), centrifuged at 12,000rpm for 15min at 4 C to remove insoluble debris. The fusion protein in the lysate was affinity-purified by Ni-NTA agarose (QIAGEN) capturing His tags after 3h incubation at 4 C. After washing with TBS supplemented with 30mM imidazole for three times, the fusion proteins were eluted by TBS supplemented with 300mM imidazole for 1h at 4 C. Purity of recombinant proteins was verified by 8% SDS-PAGE.
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!