B per
Manufactured by Thermo Fisher Scientific
Sourced in United States
B-PER is a lysis reagent designed for the rapid and efficient extraction of recombinant proteins from bacterial cells. It is a proprietary buffer formulation that helps to disrupt cell membranes and release soluble proteins without the need for mechanical disruption.
Automatically generated - may contain errors
Lab products found in correlation
Imidazole, by Merck Group (4 mentions)
Hispur ni nta resin, by Thermo Fisher Scientific (3 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Ampicillin, by Merck Group (3 mentions)
Ni nta resin, by Qiagen (3 mentions)
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Y per, by Thermo Fisher Scientific (2 mentions)
Isopropyl β d thiogalactopyranoside, by Merck Group (2 mentions)
Tbbpa derivatives, by AccuStandard (2 mentions)
Nunc maxisorp flat bottom 96 well plate, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Pierce universal nuclease, by Thermo Fisher Scientific (2 mentions)
Hispur cobalt resin, by Thermo Fisher Scientific (2 mentions)
Benzamidine, by Merck Group (2 mentions)
Las 3000 imager, by Fujifilm (2 mentions)
T7 express e coli cells, by New England Biolabs (2 mentions)
Profinity imac resin, by Bio-Rad (2 mentions)
75 protocols using b per
1
Purification of Salmonella Pdu Bacterial Microcompartments
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S. enterica Pdu BMCs were expressed and purified from an E. coli host R995+PduST (57 (link)). Briefly, A single colony of R995+PduST was used to inoculate 100 mL of 2× YT supplemented with kanamycin and 0.5% 1,2-propanediol and grown overnight at 37°C for ~16 h. The next day, the culture was pelleted; resuspended in 20 mL of a solution containing 50 mM Tris-HCl (pH 8.0), 200 mM KCl, 5 mM MgCl2, 5 mM β-mercaptoethanol (βME), 0.5 mM EDTA, 0.5 mg/mL lysozyme, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 50% (vol/vol) B-PER (Thermo); gently rocked at room temperature (RT) for 25 min; and then placed on ice for 5 min. Genomic DNA was fragmented by two 1-s pulses from a sonicator. Insoluble matter was pelleted for 20 min at 13,000 × g at 4°C. BMCs were pelleted at 17,500 × g at 4°C for 40 min. Pelleted BMCs were then resuspended in labeling buffer (20 mM phosphate [pH 7.4], 50 mM KCl, 5 mM MgCl2) and pelleted again for 20 min. The final BMC pellet was resuspended in 1 mL of labeling buffer and stored at 4°C for <2 weeks. BMCs were quantified via a Bradford assay (58 (link)) against a BSA standard.
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2
Recombinant Protein Expression and Purification
3
Recombinant Protein Expression and Purification
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The expression plasmids pGE-hRID(2m)-6xhis-EGFP (hRID(2m)-EGFP) and pGE-LysRS-6xhis-SPARC (RS-SPARC), were transformed into the BL21(DE3)pLysE and BL21(DE3)pLysS E. coli strains, respectively. A single colony was inoculated into 3 mL Luria-Bertani (LB) medium containing 50 µg/mL ampicillin and 34 µg/mL chloramphenicol, then cultured overnight at 37 °C. Approximately 0.5 mL of the overnight cultured cells were transferred to 20 mL of fresh LB medium with the same concentration of antibiotics, then cultured at 37 °C until an optical density (OD)600 of 0.5 was reached. The expression of hRID(2m)-EGFP or RS-SPARC was then induced by adding 1 mM Isopropyl beat-D-1-thiogalactopyranoside (IPTG) followed by incubation for 3 h at 37 °C or 5 h at 27 °C. Approximately 10 mL of cultured cells were harvested and suspended in 0.3 mL of the mild lysis buffer B-PER (Thermo Scientific, 90078, Rockford, IL, USA). The suspended cells were sonicated or incubated at room temperature, and separated into total, soluble, and insoluble fractions by centrifugation at 15,000× g for 12 min at 4 °C. The expression of target proteins was analyzed by SDS-PAGE. The fluorescent protein bands and Coomassie Blue stained protein gels were imaged using a gel documentation system (InGenius3, SYNGENE, Frederick, MD, USA).
4
Purification of Bacterial Microcompartments
5
Recombinant Expression of ZIKV NS3 Helicase
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The ZIKV NS3
helicase coding region (1342 bp) corresponding to PDB ID: 5GJC was synthesized
by GeneArt Gene synthesis services provided by Invitrogen (USA). This
gene was further ligated into pET 151/D-TOPO vector purchased from
Thermo Fisher Scientific (USA). The final construct containing N-terminal
6X-His tag with the TEV protease cleavage site was transformed into
BL21 (Sigma) E. coli cells, and thereafter,
positive clones were expressed in LB broth media (inducing with 1
mM IPTG at 20 °C overnight). Cells containing recombinant protein
were harvested by centrifugation at 6000g at 4 °C
and re-suspended in binding buffer (50 mM Tris, 300 mM NaCl, 40 mM
imidazole, 5% glycerol pH 8.0). Protease inhibitor cocktail (Thermo
Fisher Scientific, USA) was added before cell lysis. Cells were lysed
by sonication and adding 50% B-PER (Thermo scientific) reagent. After
centrifugation at 16,000 rpm for 30 min at 4 °C, the supernatant
containing recombinant protein was filtered and loaded on a HisTrap
FF 5 mL column (GE healthcare). Recombinant protein was eluted by
using a linear imidazole gradient from 0 to 100%. Eluted fractions
were analyzed on 10% SDS-PAGE for purity. Buffer exchange was carried
out to remove the Imidazole by using Amicon 10 kDa (Merck Millipore)
centrifugal filters. Recombinant protein was kept in storage buffer
(50 mM Tris, 100 mM NaCl, 5% glycerol) stored in aliquots at −80
°C.
helicase coding region (1342 bp) corresponding to PDB ID: 5GJC was synthesized
by GeneArt Gene synthesis services provided by Invitrogen (USA). This
gene was further ligated into pET 151/D-TOPO vector purchased from
Thermo Fisher Scientific (USA). The final construct containing N-terminal
6X-His tag with the TEV protease cleavage site was transformed into
BL21 (Sigma) E. coli cells, and thereafter,
positive clones were expressed in LB broth media (inducing with 1
mM IPTG at 20 °C overnight). Cells containing recombinant protein
were harvested by centrifugation at 6000g at 4 °C
and re-suspended in binding buffer (50 mM Tris, 300 mM NaCl, 40 mM
imidazole, 5% glycerol pH 8.0). Protease inhibitor cocktail (Thermo
Fisher Scientific, USA) was added before cell lysis. Cells were lysed
by sonication and adding 50% B-PER (Thermo scientific) reagent. After
centrifugation at 16,000 rpm for 30 min at 4 °C, the supernatant
containing recombinant protein was filtered and loaded on a HisTrap
FF 5 mL column (GE healthcare). Recombinant protein was eluted by
using a linear imidazole gradient from 0 to 100%. Eluted fractions
were analyzed on 10% SDS-PAGE for purity. Buffer exchange was carried
out to remove the Imidazole by using Amicon 10 kDa (Merck Millipore)
centrifugal filters. Recombinant protein was kept in storage buffer
(50 mM Tris, 100 mM NaCl, 5% glycerol) stored in aliquots at −80
°C.
6
Immunoblotting of Bacterial Proteins
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We pelleted cells from each condition at 5,000 g for 10 min. After the supernatant was decanted, the pellets were resuspended in 60 μl of B‐PER (Thermo Scientific) containing complete Mini ethylenediaminetetraacetic‐acid‐free protease inhibitor cocktail (Roche), and cells were lysed for 30 min at room temperature. After pelleting the cell debris by centrifugation at 15,000 g for 5 min, we transferred 40 μl of clear lysate to a new tube. We determined protein concentrations using a Bradford assay reagent (Thermo Scientific) and denatured proteins by adding 10% sodium dodecyl sulfate [SDS] (Sigma‐Aldrich) to a final concentration of 2%. Next, 60 μg of total protein from each cell lysate were loaded and separated by 12% SDS‐polyacrylamide gel electrophoresis. The proteins were transferred onto a polyvinylidene fluoride membrane (Immobilon, 0.45 μm, Millipore) and probed via immunoblotting. We used the following antibodies for immunoblotting: rabbit anti‐TcpA (Taylor et al, 2004 (link); 1:2,500), mouse anti‐RnaP (Biolegend; 1:2,500), anti‐rabbit horseradish peroxidase‐conjugated (Promega, 1:2,500), and anti‐mouse horseradish peroxidase‐conjugated (Invitrogen; 1:2,500). The immunoblots were developed with the SuperSignal West Pico chemiluminescent kit (Pierce).
7
Recombinant SARS-CoV-2 Spike and Nucleocapsid Proteins
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Synthetic nucleic acids encoding the SARS-CoV-2 S-RBD (amino acids 319–516) or full-length N protein were codon optimized for expression in E. coli and ordered as gblock gene fragments (IDTDNA). Fragments were cloned into a pRSET A bacterial expression plasmid (Invitrogen) utilizing BamHI/HindIII. Plasmids were verified by restriction digest and Sanger sequencing. Plasmids were used to transform BL21(DE3) E. coli (NEB). Protein was induced by autoinduction [79 (link)]. Bacteria were lysed using bacterial protein extraction reagent (BPER) (Thermo Fisher) with HALT protease inhibitors (Thermo Fisher). Insoluble material including protein of interest was pelleted spinning at 10,000×g for 10 min. Insoluble pellets were washed twice in inclusion body wash buffer (20 mM Tris–HCl, pH7.5, 10 mM EDTA, 1% triton X-100), resolubilized using 50 mM CAPS, pH 11.0, 1% N-lauroylsarcosine, and 1 mM dithiothreitol (DTT) with end-over-end mixing at room temperature for 30 min. Soluble protein was dialyzed 3 × against 20 mM Tris-HCl, pH 8.5 with 0.1 mM DTT [2 ]. Dialyzed proteins were purified by nickel affinity chromatography (Hispur kit, Thermo Fisher 88229). Purified proteins were denatured by boiling in Laemmli sample buffer, separated by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining. Proteins were stored at − 80 until use.
8
Affinity Purification of Ribosome Biogenesis Proteins
9
Protein Extraction and Western Blot Analysis of M. avium
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M. avium subsp. hominissuis isolates were cultured for 2 weeks in 7H9 broth. Once turbid, the cultures were centrifuged at 5,000 × g for 10 min, supernatant was removed, and pellets were stored at –80°C. Extraction of M. avium subsp. hominissuis total proteins was performed using B-PER (Thermo Fisher Scientific, Waltham, MA) supplemented with DNase I and lysozyme (Thermo Fisher Scientific). Twenty micrograms of M. avium subsp. hominissuis total proteins and Mycobacterium tuberculosis strain H37Rv culture filtrate proteins (BEI Resources NR-14825) was separated by SDS-PAGE on a 4 to 12% Bis-Tris–morpholineethanesulfonic acid (MES) gel (Thermo Fisher Scientific) and transferred onto a polyvinylidene difluoride (PVDF) membrane (iBlot, Invitrogen). Membranes were blocked in 5% nonfat milk-PBS with 0.25% Tween 20 (PBST) overnight at 4°C. Polyclonal anti-LAM primary antibody (BEI Resources NR-13821) was diluted 1:1,000 in 5% nonfat milk in PBST and used with 1:1,000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific). NR-14825 and 13821 reagents were obtained through BEI Resources, NIAID, and the NIH. Blots were visualized using SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific) on the iBright CL1000 imager (Invitrogen, Carlsbad, CA).
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10
Purification of Cry3Bb1 Protein Variants
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N-terminal His-tagged Cry3Bb1 variants and wild type Cry3Bb1 were expressed in E. coli Rosetta2 (DE3) cells using standard growth conditions for lac promoter induction in media with 1 mM IPTG and 100 ug/ml kanamycin and 25 µg/ml chloramphenicol and incubated at 18 °C for 48 hours. Cells were pelleted and lysed with a 3:1 mixture of B-PER and Y-PER (Thermo Fisher Scientific; Waltham, MA), 25 mM Tris-HCl pH 8, supplemented with 150 mM NaCl, 0.1 mg/ml lysozyme and EDTA free protease inhibitor cocktail (MilliporeSigma; Burlington, MA). Cleared lysate was incubated with His-Select Nickel Affinity gel (MilliporeSigma; Burlington, MA) at 4 °C for 30 min. The affinity gel was washed in buffer containing 250 mM NaCl, 30 mM Tris-HCl pH 8.0, 10 mM imidazole and eluted in a buffer of 250 mM NaCl, 30 mM Tris-HCl pH 8.0, and 200 mM imidazole. Further purification of eluted proteins was conducted using a HiTrap Q HP column (GE Healthcare) using a salt gradient from buffer A containing 50 mM sodium carbonate pH 9.0 to buffer B containing 50 mM sodium carbonate pH 9.0 and 1M NaCl, with a final elution resulting in approximately 200mM NaCl.
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