P. gingivalis W83, P. gingivalis ATCC 33 277 and P. gulae ATCC 51 700 (ATCC, VA) bacteria were grown in modified tryptic soy broth media (TSB media supplemented with 5 mg mL−1 yeast extract, 5 μg mL−1 hemin, 1 μg mL−1 menadione, 0.5 mg mL−1 L‐cysteine) to late mid‐log phase, collected by centrifugation for 20 minutes at 4000 g, washed in PBS, then resuspended in PBS. Fifty microliters containing approximately 106 CFUs bacteria was used for each Kgp activity assay, for potency determination from intact bacteria. To prepare lysates for analysis, bacteria were washed in PBS and lysed in B‐Per lysis buffer (Thermo Fisher Scientific) on ice for 20 minutes, centrifuged for 30 minutes at 16 000g, 4°C, and supernatant lysates collected. Protein concentration was measured using Pierce BCA protein assay kit (Thermo Fisher). When lysates were used for Kgp activity assays, 500 ng of bacterial lysate was used for the assay.
B per lysis buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
B-PER lysis buffer is a product designed for the efficient extraction of proteins from bacterial cells. It is a ready-to-use solution that effectively lyses the bacterial cell wall and membrane, releasing the target proteins. The buffer is formulated to maintain the functional integrity of the extracted proteins.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Gst bulk kit, by GE Healthcare (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Dynabeads his tag isolation and pulldown, by Thermo Fisher Scientific (1 mentions)
Pierce glutathione magnetic agarose beads, by Thermo Fisher Scientific (1 mentions)
6 protocols using b per lysis buffer
1
Gingipain Activity Assay of Porphyromonas
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P. gingivalis W83, P. gingivalis ATCC 33 277 and P. gulae ATCC 51 700 (ATCC, VA) bacteria were grown in modified tryptic soy broth media (TSB media supplemented with 5 mg mL−1 yeast extract, 5 μg mL−1 hemin, 1 μg mL−1 menadione, 0.5 mg mL−1 L‐cysteine) to late mid‐log phase, collected by centrifugation for 20 minutes at 4000 g, washed in PBS, then resuspended in PBS. Fifty microliters containing approximately 106 CFUs bacteria was used for each Kgp activity assay, for potency determination from intact bacteria. To prepare lysates for analysis, bacteria were washed in PBS and lysed in B‐Per lysis buffer (Thermo Fisher Scientific) on ice for 20 minutes, centrifuged for 30 minutes at 16 000g, 4°C, and supernatant lysates collected. Protein concentration was measured using Pierce BCA protein assay kit (Thermo Fisher). When lysates were used for Kgp activity assays, 500 ng of bacterial lysate was used for the assay.
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P. gingivalis W83, P. gingivalis ATCC 33 277 and P. gulae ATCC 51 700 (ATCC, VA) bacteria were grown in modified tryptic soy broth media (TSB media supplemented with 5 mg mL−1 yeast extract, 5 μg mL−1 hemin, 1 μg mL−1 menadione, 0.5 mg mL−1 L‐cysteine) to late mid‐log phase, collected by centrifugation for 20 minutes at 4000 g, washed in PBS, then resuspended in PBS. Fifty microliters containing approximately 106 CFUs bacteria was used for each Kgp activity assay, for potency determination from intact bacteria. To prepare lysates for analysis, bacteria were washed in PBS and lysed in B‐Per lysis buffer (Thermo Fisher Scientific) on ice for 20 minutes, centrifuged for 30 minutes at 16 000g, 4°C, and supernatant lysates collected. Protein concentration was measured using Pierce BCA protein assay kit (Thermo Fisher). When lysates were used for Kgp activity assays, 500 ng of bacterial lysate was used for the assay.
2
Recombinant FoxO3A Protein Purification
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NEB5-alpha bacterial strains (#C2987I, New England Biolabs) transformed with different pGEX-4T3-GST-FoxO3A constructs were grown in Luria Broth medium with Ampicillin (#13399, Sigma) and induced with 0.5 mM IPTG when they reached the optical density of 0.6 (A600) at 37 °C, for 4 h. Cells were then collected by centrifugation and pellets were lysed with B-PER lysis buffer (#78248, Thermo Fisher Scientific). The lysate was centrifuged at 15,000 × g for 5 min at 4 °C. Recombinant protein expression was confirmed by SDS-PAGE. Fusion proteins were purified by affinity chromatography using the GST Bulk Kit (#27-4570-01, GE Healthcare, Milwaukee, WI) according to the manufacturer’s instructions. GST-fused proteins were evaluated and quantified by SDS-PAGE.
3
Bacterial Cell Lysis and Protein Extraction
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Bacteria (108) were collected and centrifuged at 5000g for 10 min at 4°C. The supernatant was discarded. Bacterial cell pellet was lysed with 1 ml of B-PER lysis buffer (Thermo Fisher Scientific) on ice for 10 min and then centrifuged for 10 min at 14,000g at 4°C. The supernatant containing protein lysate was collected.
4
Recombinant Expression and Purification of Arc Proteins
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To express and purify Arc proteins in the E. coli system, the cDNAs of dArc1, dArc2 and rArc were subcloned into the pGEX-4T-1 vectors together with GST-6xHis-tags and TEV protease cleavage site. Arc proteins were expressed in E. coli strain BL21 (DE3) grown in LB broth by induction of log-phase cultures with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated overnight at 23 °C. Cells were pelleted and resuspended in B-PER lysis buffer (#78243, Thermo Fisher Scientific) before centrifugation to collect cell lysates.
Tagged Arc proteins were pulled down using Ni-NTA resin column and the eluates with GST-6xHis-dArc1 and GST-6xHis–rArc proteins were used for peptide binding assays.
Tagged Arc proteins were pulled down using Ni-NTA resin column and the eluates with GST-6xHis-dArc1 and GST-6xHis–rArc proteins were used for peptide binding assays.
5
Recombinant Protein Purification from E. coli
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BL21 competent cells, transformed with different constructs, were grown in LB medium with antibiotics and induced with IPTG. Cells were lysed with B-PER lysis buffer (#78248, Thermo Fisher Scientific). GST-fusion proteins were purified with Pierce Glutathione Magnetic Agarose Beads (TH269836, Thermo Fisher Scientific) according to the manufacturer’s instructions. His-fusion proteins were purified with Dynabeads His-Tag Isolation and Pulldown (10104D, Thermo Fisher Scientific) according to the manufacturer’s instructions.
6
Recombinant Human TRAIL Production
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RhTRAIL was produced according to the method established previously [18] (link), [19] (link). Briefly, we use a M15 pRep4 bacteria with pQE9 expression plasmid (Qiagen), which could code for the sequence MRGSHHHHHHGSEQKLISEEDLNLQ (His6-Myc) followed by amino acids 95-281 of human TRAIL [20] (link). When growing to the density of 0.7 at 600 nm, bacteria were induced with 50 µg/ml isopropyl-1-thio-β-D-galactopyranoside (IPTG). After 18 h at 37°C, the bacteria was suspended with B-PER lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's guidance. The filtered supernatant was washed after centrifugation with 2 column volumes of TBS (10 Mm Tris-HCL pH 7.4, 140 mM NaCl) containing 10 mM imidazole followed by 2 column volumes of TBS with 50 mM imidazole. After that, the production was eluted with TBS, containing 0.5 mM imidazole, and dialyzed in a dialysis bag. After confirmed by SDS-PAGE, rhTRAIL was stored at -80°C and was used within 8 months to assure its effectiveness.
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