LPS from V. cholerae O1 was extracted using an LPS extraction kit (iNtRON Biotechnology, Seongnam, Korea), according to the manufacturer’s instructions. LPS from V. cholerae O1 was extracted using an LPS extraction kit (iNtRON Biotechnology, Seongnam, Korea), according to the manufacturer’s instructions. Briefly, bacterial cultures were centrifuged and re-suspended in lysis buffer and vortexed vigorously to dissolve cell clumps. Then, the suspension was mixed with chloroform and centrifuged at 13,000 rpm for 10 min at 4 °C. Then, the aqueous layer was mixed with purification buffer and was centrifuged. The pellet was washed with 70% ethanol and centrifuged at 13,000 rpm for 3 min at 4 °C. The pellet was dried at room temperature and dissolved in 10 mM Tris–HCl (pH 8.0) by boiling for 2 min. The extracted LPS was fractionated using SDS‐PAGE. The SDS-PAGE gel was then submitted to silver staining (Additional file 1 : Fig. S1). The silver-stained SDS-PAGE of the LPS extract demonstrated two bands of molecular weights ~ 35 and ~ 15 kDa, corresponding to antigen-O and lipid A core, respectively (Additional file 1 : Fig. S1). For the in vivo experiment, the extracted LPS was detoxified (DLPS) by alkaline treatment, as described previously [27 (link)].
Lps extraction kit
Manufactured by iNtRON Biotechnology
Sourced in United States, Cameroon, Japan
The LPS extraction kit is a laboratory product designed to isolate and extract lipopolysaccharides (LPS) from bacterial samples. The kit provides the necessary reagents and protocols to perform this extraction process effectively.
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Lab products found in correlation
Ready gel precast tris hcl polyacrylamide gels, by Bio-Rad (2 mentions)
Periodate, by Merck Group (2 mentions)
Periodate, by Solarbio (2 mentions)
Escherichia coli o111 b4, by Merck Group (2 mentions)
Sodium acetate, by Merck Group (2 mentions)
Proteinase k, by Solarbio (2 mentions)
Limulus amebocyte lysate chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (2 mentions)
Escherichia coli 0111b4 lps, by Thermo Fisher Scientific (2 mentions)
Anaerobic chamber, by Coy Laboratory Products (2 mentions)
Odyssey two color infrared imaging system, by LI COR (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Irdye680cw conjugated donkey anti mouse igg polyclonal antibody, by LI COR (2 mentions)
Fugene hd, by Promega (1 mentions)
Silver staining kit, by Bio-Rad (1 mentions)
Silver stain kit, by Bio-Rad (1 mentions)
Limulus amebocyte lysate kit, by Lonza (1 mentions)
E coli lps, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Gel imager, by Bio-Rad (1 mentions)
Pro q emerald 300 lipopolysaccharide gel stain, by Thermo Fisher Scientific (1 mentions)
53 protocols using lps extraction kit
1
Vibrio cholerae O1 LPS Extraction
2
MALDI-TOF Analysis of F. tularensis LPS
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LPS from F. tularensis strains was prepared using a LPS extraction kit (Catalog # 17141) from Intron Biotechnology. Sterility of the LPS preparations was confirmed. The samples were analyzed by matrix-assisted laser desportion ionization time-of-flight (MALDI-TOF) mass spectrometry analysis using protocols developed by Zhou et al [79 (link)]. In short, 20 μl of each LPS sample was mixed with 80 μl of methanol/chloroform in a glass vial, briefly vortexed and 1 μl of the solubilized sample spotted on a stainless steel target. Samples were allowed to air dry and 0.5 μl of matrix (10 mg/ml 2,5-dihydrobenzoic acid) was added to each spot. Samples were analyzed by MALDI-TOF mass spectrometry in reflector/negative ion mode using an Applied Biosystems 5800 instrument (Foster City, CA). The instrument was calibrated with low molecular weight standards (Bruker; Billerica, MA) and data were collected from 800 to 4000 (m/z) by manual “hot spot” searching and adjusting laser intensity to obtain optimum signal to noise for each sample. Each of the reported spectra is averages of 1000 laser shots.
3
LPS Extraction and Transfection Protocol
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LPS was extracted from purified OMV using LPS extraction kit (iNtRON Biotechnology) according to manufacturer's instruction. LPS concentration was determined by Purpald assay as described in (Lee and Tsai, 1999) . Transfection of cells with purified LPS was done at a concentration of 500ng/50,000 cells, using FuGeneHD (Promega) transfection reagent in Opti-MEM, as previously described (Santos et al., 2018) .
4
Lipid A Isolation from Subgingival Samples
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LPS from subgingival samples was extracted using the LPS extraction kit (iNtRON Biotechnology, S.Korea) following the manufacturer's instructions. Extracted LPS was re-suspended in 500µl of LPS-free water and stored at 4 o C. For lipid A isolation, all healthy, diseased and post-treatment samples were pooled and lipid A was isolated by mild hydrolysis as described by Coats et al. [13] .
5
Lipopolysaccharide Extraction and Phage Inactivation
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LPS extraction from A. xylosoxidans was performed using an LPS extraction kit from Intron Biotechnology (17144; Boca Scientific, Boca Raton, FL, USA), according to the manufacturer’s instructions. LPS from Escherichia coli O111:B4 purchased from Sigma-Aldrich, Inc. (L2630; Sigma, USA) was used as a negative control to ensure that the possible effect was specific to A. xylosoxidans LPS. Both LPS of Escherichia coli O111:B4 and A. xylosoxidans A22732 are smooth type. The phage inactivation by LPS was performed as previously described34 (link).
6
Highly Pure LPS Extraction
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A 2 mL bacterial culture with an OD600 = 1 was pelleted by centrifugation and washed in 10 mm MgCl2 (to remove any trailing media). LPS was then extracted using an LPS extraction kit (iNtRON Biotechnology, Gyeonggi, South Korea). The LPS pellet was resuspended in 50 µL of 10 mm Tris, pH 8. To ensure complete solubilization of the LPS pellet, the sample was boiled at 95 °C for 2 min and was further treated with 3 µg/µL of proteinase K at 50 °C for 30 min to obtain highly pure LPS from bacterial cells.
7
Endotoxin Activity of Salivary and Subgingival LPS
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LPS from salivary and subgingival plaque samples was extracted using the LPS extraction kit (iNtRON Biotechnology, S. Korea), following the manufacturer's instructions. Extracted LPS was re-suspended in 500 µl of LPS-free water and stored at 4 °C. Endotoxin activity of salivary and subgingival LPS extracts was measured (EU/ml), in duplicates, by an endpoint, fluorescent, recombinant Factor C assay according to manufacturer's instructions (EndoZyme, Hyglos, Germany). All samples were anonymised and laboratory staff were blinded until the end of the study.
8
Extraction and Characterization of Bacterial LPS
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The LPS from colistin-resistant isolates and a colistin susceptible standard strain (E. coli strain ATCC 25922) was extracted using the LPS Extraction Kit (iNtRON Biotechnology, South Korea) according to the manufacturer’s instructions. The purity of extracted LPS was evaluated by silver staining of SDS-PAGE gels and HPLC analysis. Limulus Amebocyte Lysate (LAL) and rabbit pyrogen tests were done to evaluate the functionality of the purified LPS.
9
Lipopolysaccharide Extraction and Phage Inactivation
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LPS was extracted from A. xylosoxidans using an LPS extraction kit from Intron Biotechnology (17144; Boca Scientific, Boca Raton, FL, USA), according to the manufacturer’s instructions. A control containing LPS from Escherichia coli O111:B4, purchased from Sigma-Aldrich, Inc. (L2630; Sigma, USA), was used as the negative control to ensure that any effect was specific to A. xylosoxidans LPS. Phage inactivation by LPS was tested as previously described24 (link).
10
Efficient LPS Extraction Protocol
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LPS was extracted using an LPS extraction kit (Intron Biotechnology, Seongnam-Si, Republic of Korea) with some modifications. Briefly, cells were harvested and lysed in lysis buffer (50 mg of cells/mL of lysis buffer) and then subjected to a vigorous vortex to dissolve cell clumps. After the addition of chloroform, the sample was centrifuged for 45 min at 4 °C. The upper aqueous layer was collected, and 2 volumes of purification buffer were added to 1 volume of aqueous layer. The mixture was then incubated at −20 °C for 2 h, before centrifugation at 20,000× g for 10 min at 4 °C. The resulting LPS samples were purified to remove any remaining particulates and stored in sterile vials at 4 °C.
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