B per protein extraction reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States

The B-PER Protein Extraction Reagent is a ready-to-use solution designed for the rapid and efficient extraction of proteins from bacterial cells. It is a mild, non-denaturing detergent-based reagent that effectively lyses bacterial cells and solubilizes proteins, allowing for their subsequent purification or analysis.

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Lab products found in correlation

Hispur cobalt spin column, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4b beads, by GE Healthcare (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4b, by GE Healthcare (2 mentions) Coli rosetta2, by Merck Group (1 mentions) Codon plus, by Agilent Technologies (1 mentions) Bradford protein assay kit, by Thermo Fisher Scientific (1 mentions) Hispur ni nta agarose resin, by Thermo Fisher Scientific (1 mentions) Lysozyme, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Glutathione sepharose 4b beads, by Roche (1 mentions) Glutathione sepharose resin, by GE Healthcare (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Edta free protease inhibitor cocktail tablet, by Roche (1 mentions) Pgex 6p 1, by GE Healthcare (1 mentions) Lb medium, by Carl Roth (1 mentions) Glutathione sepharose 4b, by Thermo Fisher Scientific (1 mentions) Escherichia coli bl21 de3, by Merck Group (1 mentions) Hitrap chelating hp column, by GE Healthcare (1 mentions) Hitrap, by Cytiva (1 mentions)

Spelling variants of this product

Protein extraction reagent (96 citations) B-PER (75 citations) B-PER reagent (41 citations) B-PERTM Bacterial Protein Extraction Reagent (3 citations) Extraction Reagents (2 citations)

17 protocols using b per protein extraction reagent

Purification of N-terminal 6xHis-tagged NanA from S. pneumoniae

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In order to obtain the nanA from S.
pneumoniae strain DSM20566, primer pair NA_116aaNdeI_fw
(5’-TGCACGACATATGGAAAATGTC-3’) and
NA_Cter_XhoI_rv
(5’-TCAAATCTCGAGAATTCTTCTCT-3’) were
applied to amplify this gene. The NdeI/XhoI double-digested PCR product was then
ligated to the E. coli expression vector pET-28a. The
constructed plasmid encodes an N-terminal 6× His-tagged 886aa protein. A
150 mL E. coli culture of BL21(DE3)/pTNA20566-116aa was used to
synthesize the enzyme. Gene expression was induced by IPTG at a final
concentration of 0.5 mM after a 4 h initial cultivation at 37°C. The
broth was further incubated overnight at 25°C before harvested by
centrifugation. Cell pellet was subjected to B-PER Protein Extraction Reagents
(Thermoscientific) for lysis and protein release. N-terminal His-tagged NanA was
purified via HisPur Cobalt Spin Column (Thermoscientific) and desalted by Pierce
Concentrators PES 30K MWCO. Protein sample was stored in a 50% glycerol solution
at -20°C.

Walther E., Richter M., Xu Z., Kramer C., von Grafenstein S., Kirchmair J., Grienke U., Rollinger J.M., Liedl K.R., Slevogt H., Sauerbrei A., Saluz H.P., Pfister W, & Schmidtke M. (2014). Antipneumococcal activity of neuraminidase inhibiting artocarpin. International journal of medical microbiology : IJMM, 305(3), 289-297.

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GST-GLI1 Fusion Protein Expression

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GST-GLI1 fusion proteins were expressed in E. coli Rosetta2 (Merck Millipore) or Codon Plus (Agilent Technology). E. coli were lysed in B-PER Protein Extraction Reagents (Thermo Fisher Scientific), according to the manufacturer’s instructions. Protein extracts were centrifuged at 15,000×g for 15 min at 4 °C, and the supernatant (5 µg protein) was incubated with 20 µl glutathione sepharose 4B beads (GE Healthcare) for 2 h at 4 °C. The beads were then washed three times with PBS or methylation reaction buffer (MRE; 50 mM Tris–HCl [pH 7.5], 0.1 mM EDTA, and 50 mM NaCl).

Abe Y., Suzuki Y., Kawamura K, & Tanaka N. (2019). MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex. Communications Biology, 2, 23.

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Recombinant NanA Purification from E. coli

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A 100 mL culture of E. coli BL21(DE3) containing the NanA construct was initially incubated at 37 ^oC for 3–4 h, then IPTG was added to a final concentration of 0.5 mM for gene expression induction. The broth was further incubated overnight at 25 °C with rigorous shaking and harvested by centrifugation. The resulting cell pellet was subjected to B-PER Protein Extraction Reagents (Thermoscientific) for lysis and protein release. N-terminal His-tagged NanA was purified via HisPur Cobalt Spin Column (Thermoscientific) and desalted by Pierce Concentrators PES 30K MWCO. Purified enzyme was preserved in a 40–50% glycerol solution at −20 °C.

Xu Z., von Grafenstein S., Walther E., Fuchs J.E., Liedl K.R., Sauerbrei A, & Schmidtke M. (2016). Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility. Scientific Reports, 6, 25169.

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Expression and Purification of EGFP-CNA35 Protein

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The pET28a-EGFP-CNA35 DNA construct purchased from Addgene was described before [34] .
The procedures for expression and purification of the EGFP-CNA35 protein has been detailed in our previous publications [49] . In brief, the DNA construct was transformed into BL21(DE3) competent E. Coli, and the protein expression was induced by Isopropyl β-D-1thiogalactopyranoside (IPTG) in liquid culture at room temperature (~25ºC). After lysis of the bacterial pellets in B-Per protein extraction reagents (Thermo), the 6xHis-tagged EGFP-CNA35 protein was pulled down by HisPur Ni-NTA agarose resin (Thermo), and purified by nickel chelation chromatography. The purified protein solution was examined by 10% SDS-PAGES (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Coomassie Blue staining, which showed only one major band with molecular size of ~60 kD (Fig. S1C). The protein concentration was determined by Bradford Protein Assay Kit (Thermo).
To label COL, purified EGFP-CNA35 protein was incubated with COL solution at a mass ratio of 1:20 on ice for 10-15 min, followed by neutralization with a basic solution and further mixing with Matrigel solution for gel formation at 37ºC.

Ouyang M., Qian Z., Bu B., Jin Y., Wang J., Zhu Y., Liu L., Pan Y, & Deng L. (2020). Sensing Traction Force on the Matrix Induces Cell-Cell Distant Mechanical Communications for Self-Assembly. ACS biomaterials science & engineering, 6(10).

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Recombinant GST-AGO2 Purification

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The prokaryotic expression vector pGEX-4T-1-AGO2 was transformed into BL21 competent cells with 0.5 mM IPTG inducing for 16 h at 16 °C. Bacterial cells were lysed in B-PER Protein Extraction Reagent (#78248, Thermo Fisher, USA) with 100 μg/ml lysozyme (Sigma), 12.5 μl/ml protease inhibitor cocktails (Sangon) and 2 U/ml DNaseI (Thermo Fisher) for 1 h at room temperature. The fusion protein GST-AGO2 was purified with Glutathione sepharose 4B beads (GE healthcare) and gradually eluted with 20 mM GSH (reduced glutathione; 50 mM Tris pH8.0) and 10 mM GSH for 15 minutes. For in vitro GST-AGO2 acetylation or deacetylation assays, HEK293T cells were transfected with indicated plasmids or treatment as indicated conditions, then cells were lysed with RIPA buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche)) on ice for 1 h. And then lysates were incubated with 10 μg of GST-AGO2 and 25 μl of Glutathione sepharose 4B beads at 4 °C overnight and then incubated at 30 °C for 0.5 h. GST-AGO2 bound to beads were washed for five times by using the same RIPA buffer, and followed by Western blotting analysis through the indicated antibodies.

Zhang H., Wang Y., Dou J., Guo Y., He J., Li L., Liu X., Chen R., Deng R., Huang J., Xie R., Zhao X, & Yu J. (2018). Acetylation of AGO2 promotes cancer progression by increasing oncogenic miR-19b biogenesis. Oncogene, 38(9), 1410-1431.

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Expression and Purification of Strep-PTE

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pET-Strep-PTE plasmids were transformed into E. coli BL21 (DE3) grown for 8 h at 30 °C in Overnight Express Instant TB medium (Novagen) containing 100 µg/mL ampicillin and 200 μM ZnCl₂ before lowering the temperature to 16 °C and continuing incubation overnight. Cells were harvested by centrifugation, resuspended and lysed using a 1:1 mixture of B-PER Protein Extraction Reagent (Thermo Scientific): 50 mM Tris-HCl buffer, pH 7.5 containing 200 μM ZnCl₂, 100 μg/mL lysozyme and ~1 μL of benzonase per 100 mL. Cell debris was removed by centrifugation and the clarified lysate passed through a 45 μm filter before loading onto a Strep-Tactin Superflow High capacity column (1 mL). Strep-PTE variants were eluted with Elution buffer (100 mM Tris-HCl, pH 7.5, 200 μM ZnCl₂ and 2.5 mM desthiobiotin) according to the manufacturer’s instructions (IBA Lifesciences).

Emond S., Petek M., Kay E.J., Heames B., Devenish S.R., Tokuriki N, & Hollfelder F. (2020). Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis. Nature Communications, 11, 3469.

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Generating CG7099 Protein Fragment

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cDNA corresponding to CG7099 amino acids 1,357 to 1,907 was obtained from the Drosophila Genomics Resource Center (DGRC clone LD46862), PCR-amplified introducing a BglII restriction site upstream of the coding sequence, and subcloned into a pET-23a vector containing a glutathione-S-transferase (GST) and His tag at the carboxyl and amino termini, respectively. CG7099 protein fragment expression was induced by IPTG (0.5 mM) in BL21-CodonPlus^® Competent Cells grown to a culture density of approximately OD₆₀₀ 0.5, and shaken for approximately 100 rpm for 2 h. Cells were subsequently pelleted and proteins extracted via the B-PER protein extraction reagent (ThermoScientific product number 78243; Waltham, Massachusetts, USA). Polyclonal rabbit antibodies were generated against the isolated CG7099 fragment at the Pocono Rabbit Farm and Laboratory (Canadensis, Pennsylvania, USA). Quality control and antigen specificity were tested by peptide competition assays against Kc167 lysate with rabbit polyclonal α-dTFIIIC220 antibody pre-incubated with bacterial extract expressing GST empty construct or GST-CG7099 construct expressing a fragment corresponding to amino acids 1,357 to 1,907 (Additional file 1).

Van Bortle K., Nichols M.H., Li L., Ong C.T., Takenaka N., Qin Z.S, & Corces V.G. (2014). Insulator function and topological domain border strength scale with architectural protein occupancy. Genome Biology, 15(5), R82.

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Purification of GST-MESP-1 and GST

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GST–MESP-1 and GST were cloned into pGEX 6P-1 (GE) and expressed in BL21 Escherichia coli cells. Cultures were grown at 37ºC to an OD of 0.6–0.8 and then induced at 28ºC for 4 h with 0.1 mM IPTG. After harvesting, cells were lysed at room temperature for 15 min with B-PER Protein Extraction Reagent (ThermoFisher Scientific, Waltham, MA) including 100 μg/ml lysozyme, 20 μl DNase I (ThermoFisher), and EDTA-free protease inhibitor cocktail tablets (Roche, Indianapolis, IN). The lysate was then cleared by centrifugation at 40,000 rpm for 35 min. Lysate was applied to glutathione Sepharose resin (GE) and then washed with wash buffer (phosphate-buffered saline, pH 7.4, 250 mM NaCl, 0.1% Tween 20, 2 mM benzamidine-HCl, 1 mM dithiothreitol [DTT]). Bound protein was then eluted with elution buffer (50 mM Tris, pH 8.1, 75 mM KCl, 10 mM reduced glutathione). Protein was dialyzed into BRB80 (80 mM 1,4-piperazinediethanesulfonic acid [PIPES], pH 6.8, 1 mM ethylene glycol tetraacetic acid [EGTA], 1 mM MgCl₂) overnight and stored at −80ºC. For all recombinant protein purifications, purity was confirmed with SDS–PAGE and concentration determined by Bradford assay (Bio-Rad, Hercules, CA).

Wolff I.D., Tran M.V., Mullen T.J., Villeneuve A.M, & Wignall S.M. (2016). Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1. Molecular Biology of the Cell, 27(20), 3122-3131.

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Analyzing KHSRP SUMOylation in Cells and In Vitro

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(A) SUMOylation of KHSRP was analyzed in 293T cells by using the method of Ni²⁺-NTA beads with His-tagged SUMO1, as described previously [29 (link), 50 (link), 51 (link)]. (B) In vitro SUMOylation assay in E.coli system with pE1E2SUMO1 was performed as previously described [20 (link), 29 (link)]. Briefly, pGEX-4T-1-KHSRP-WT was co-expressed with or without pE1E2SUMO1 plasmid in E.coli BL21 (DE3) respectively, and then lysed by using B-PER Protein Extraction Reagent (#78248, Thermo Fisher, USA) and incubated with Glutathione sepharose 4B (GE healthcare) at 4 °C overnight. The beads bound proteins were washed for three times with lysis buffer, and subjected to Western-bot for analysis of SUMO1-modified GST-KHSRP. (C). The method of immunoprecipitation (IP) was also used to detect the SUMO1 modification of endogenous KHSRP. Briefly, 293T cells were lysed in NEM-RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 20 mM N-ethylmaleimide and one complete protease inhibitor cocktail). Cell lysates (1 mg) were used for immunoprecipitation. To detect the endogenous SUMO1-KHSRP in 293T cells, 5 μl of KHSRP antibody or normal IgG (as a control) was used for immunoprecipitation. Tissues were lysed in NEM-RIPA with 0.1% SDS and 5 mM EDTA, which was according to our previous study [28 (link)] and SUMO1 antibody was used for immunoprecipitation.

Yuan H., Deng R., Zhao X., Chen R., Hou G., Zhang H., Wang Y., Xu M., Jiang B, & Yu J. (2017). SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis. Molecular Cancer, 16, 157.

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Protein Expression and Extraction Protocol

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Example 6

In preparation for protein expression at a volume of 50 mL, a preculture of 5 mL LB medium (Carl Roth GmbH, Karlsruhe) was first treated with the corresponding antibiotic and cells of each strain were taken from the agar plate and transferred into the preculture by means of an inoculating loop The preculture was then incubated for 16 h at 37° C. and 150 rpm. The main culture was taken from the preculture and inoculated with 50 mL LB medium (Carl Roth GmbH, Karlsruhe) and the corresponding antibiotic, so that the optical density at 600 nm as 0.1. The main culture was then incubated at 37° C. and 150 rpm until an optical density of 0.4-0.8 at 600 nm was reached. At this time 1 mM of isopropyl-β-D-thiogalactopyranoside was added for inducing the protein expression and the culture was incubated at 22° C. for an additional 16 h. The main culture was then centrifuged at 10,000 rpm for 10 min in order to obtain the cell pellet and then to be able to perform the protein extraction and cleaning. To this end, the cell disruption was first performed using the B-PER protein extraction reagent (Thermo Fisher Scientific, Bonn) according to the manufacturers instructions. The cell lysate thus obtained was then either used directly or processed by means of a 1 mL HisPur Ni-NTA chromatography column (Thermo Fisher Scientific, Bonn) according to the manufacturers instructions.

US10227620B2. Method for biotechnological production of methylized cinnamic acids and cinnamic acid esters, methylized phenethylamines and the coupling products thereof, particularly of cinnamic acid amides (2019-03-12). SYMRISE AG [DE]. Inventors: Gerhard Krammer [DE], Jakob Peter Ley [DE], Katrin Geißler [DE], Torsten Geißler [DE], Frauke Gomoll [DE], Peter Welters [DE], Guido Jach [DE], Ludger Wessjohann [DE].

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