Bugbuster reagent

Manufactured by Merck Group

Sourced in United States, Germany

BugBuster reagent is a specialized laboratory product designed for cell lysis and protein extraction. It is a non-enzymatic, non-detergent-based buffer solution that effectively disrupts bacterial and eukaryotic cells, releasing their contents for further analysis or purification. The core function of BugBuster reagent is to facilitate the extraction of cellular components, such as proteins, from a wide range of microbial and eukaryotic samples.

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Lab products found in correlation

Nickel affinity resin, by Thermo Fisher Scientific (2 mentions) Amicon ultra centrifugal filter unit, by Merck Group (2 mentions) Dnase 1, by New England Biolabs (2 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions) Protein assay kit, by Bio-Rad (1 mentions) Ni nta agarose, by Qiagen (1 mentions) Nickel column chromatography, by Qiagen (1 mentions) Ampicillin, by GoldBio (1 mentions) Butanone, by Merck Group (1 mentions) Acetoin, by Merck Group (1 mentions) L arabinose, by GoldBio (1 mentions) Gibson assembly kit, by New England Biolabs (1 mentions) Reduced nadph, by Merck Group (1 mentions) Taq polymerase, by Roche (1 mentions) Phusion polymerase, by Thermo Fisher Scientific (1 mentions) Talon metal affinity resin, by Takara Bio (1 mentions) Acetone, by Merck Group (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Restriction enzyme, by New England Biolabs (1 mentions) Oligonucleotide, by Integrated DNA Technologies (1 mentions)

Close spelling variants of this product

BugBuster (71 citations) BugBuster Protein Extraction Reagent (64 citations) BugBuster HT Protein Extraction Reagent (5 citations) Bugbuster 10× protein extraction reagent (2 citations)

20 protocols using bugbuster reagent

Site-directed and Random Mutagenesis of LAAO/MOG

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Mutations were introduced by PCR using expression plasmid pET15b‐laao/mog as a template and the QuikChange kit (Stratagene, La Jolla, CA, USA) using primers P1 to P10 (Table S1). For site‐directed mutagenesis, both a direct and a reverse primer were designed complementary to opposite strands of the same DNA region. The mutated genes were completely sequenced using an ABI PRISM 3500 genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA) to ensure that only the desired mutations were introduced. Each colony was cultivated in a 96‐well plate, and crude extracts were prepared using BugBuster reagent (Merck Millipore, Billerica, MA, USA).
Random mutagenesis was introduced using primers P11 and P12 (Table S1), in which target amino acid positions were replaced with a degenerate codon, NNS (N = ATGC, S = GC). Each colony was cultivated in a 96‐well plate, and crude extracts were prepared using BugBuster reagent (Merck Millipore). We screened 384 colonies (4 plates), and the library size was more than 10 times larger than the number of possible 32 cases by the NNS codon.

Im D., Matsui D., Arakawa T., Isobe K., Asano Y, & Fushinobu S. (2018). Ligand complex structures of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change. FEBS Open Bio, 8(3), 314-324.

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Studying P. abyssi PolII Intein Splicing

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To study the protein splicing and N-terminal cleavage reactions of the P. abyssi PolII intein, we used plasmid pMIH, the construction of which was previously described (and previously called pPabPolWT).^{3 (link)} Overexpression produces MIH, an in-frame fusion protein consisting of E. coli Maltose binding protein (MBP), the seven C-terminal residues of the N-extein, the 185 residue intein, the five N-terminal residues of the C-extein, and a His₆ tag. To study isolated N-terminal cleavage, we generated pMIHQACA, in which both Cys+1 and Gln185 are changed to Ala to prevent the second and third step of splicing, respectively. The UniProt accession ID for the intein is Q9V2F4. Site-directed mutagenesis was used to create mutations to residues in the EXH region using appropriate oligonucleotide primers, as verified by DNA sequencing (Psomagen, Inc).
Proteins were expressed in E. coli BL21(DE3) by induction with 1 mM isopropyl-1-β-d-thiogalactopryanoside (IPTG) of a mid-log phase culture and incubation at 20°C for 16 h. Cell pellets were resuspended in buffer A (20 mM HEPES, pH 7.5, 500 mM NaCl) and disrupted with BugBuster reagent (EMD Millipore). Proteins were purified using Talon metal affinity resin and exchanged into buffer A as previously described.^{9 (link)}

Chiarolanzio K.C., Pusztay J.M., Chavez A., Zhao J., Xie J., Wang C, & Mills K.V. (2020). Allosteric influence of extremophile hairpin motif mutations on the protein splicing activity of a hyperthermophilic intein. Biochemistry, 59(26), 2459-2467.

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Protein Expression and Purification

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pET32b(+) and pET19b(+) vectors, E. coli Rosetta-gami B (DE3)pLysS, BugBuster™ reagent and u-MAC™ cartridges were purchased from Merck KGaA (Darmstadt, Germany). A synthetic 44-residue peptide corresponding to the PMV prosegment (ENKIDNVGKKIENVGKKIGDMENKNDNVENKNDNVGNKNDNVKN) was purchased from GenicBio (Shanghai, China). A quenched fluorescent synthetic peptide substrate (HRPII; 4-(4-dimethylaminophenyl) diazenylbenzoic acid (DABCYL)-LNKRLLHETQ-E(5-[(2-Aminoethyl)amino]naphthalene-1-sulfonic acid) (EDANS), and L→A mutant HRPII DABCYL-LNKRLAHETQ-E(EDANS), was purchased from CanPeptide (Pointe-Claire, QC, Canada). All other chemicals and media were obtained from Fisher Scientific Canada (Nepean, ON, Canada) or Sigma–Aldrich (St. Louis, MO, USA).

Xiao H., Bryksa B.C., Bhaumik P., Gustchina A., Kiso Y., Yao S.Q., Wlodawer A, & Yada R.Y. (2014). The zymogen of plasmepsin V from Plasmodium falciparum is enzymatically active. Molecular and biochemical parasitology, 197(1-2), 56-63.

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Heterologous Protein Expression and Purification

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The bacterial cells containing plasmids were grown overnight at 37 °C, 250 rpm in 5 mL of LB broth containing 100 μg/mL ampicillin. This culture was inoculated into 500 mL of TB broth containing 100 μg/mL ampicillin and incubated at 37 °C, 250 rpm until the OD600 reached 1.0. The culture medium was cooled in an ice-water bath for over 1 h and, subsequently, isopropyl

β

-D-1-thiogalactopyranoside (IPTG) was added to the culture medium at the final concentration of 0.1 mM. After incubating 24–48 h at 16 °C, the cells were harvested through centrifugation at 7000× g for 10 min and were lysed in 10 mL of BugBuster reagent (EMD Millipore, Burlington, MA, USA). The fusion proteins were purified using a column of Ni-NTA agarose (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The recombinant proteins were subject to 4–20% SDS-PAGE and confirmed with Coomassie brilliant blue staining. The proteins were finally dialyzed against 1X phosphate-buffered saline (PBS, pH 7.4) and the concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).

Hersh J., Yang Y.P., Roberts E., Bilbao D., Tao W., Pollack A., Daunert S, & Deo S.K. (2023). Targeted Bioluminescent Imaging of Pancreatic Ductal Adenocarcinoma Using Nanocarrier-Complexed EGFR-Binding Affibody–Gaussia Luciferase Fusion Protein. Pharmaceutics, 15(7), 1976.

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Purification of Bacterial dUTPase Enzyme

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Wt nuclear dUTPase and mutants were expressed in E. coli BL21(DE3)RIPL (New England Biolabs Inc., Beverly, MA) for 1.5 h at 37°C using 1 mM isopropyl beta‐d‐1‐thiogalactopyranoside (IPTG). The cells were harvested at 6000g for 20 min at 4°C. The pellet was washed with 10 mL of PBS then frozen at −80°C overnight. The cells were suspended in Bugbuster reagent (EMD Millipore, Billerica, MA) where the standard company protocol was followed. Nickel column chromatography (Qiagen, Valencia, CA) was used for purification following standard company protocol. Briefly, the column was equilibrated and washed with “Buffer 1” consisting of 50 mM sodium phosphate pH 7.4, 300 mM NaCl, and 10 mM imidazole. The protein was eluted with “Buffer 2″ containing 50 mM sodium phosphate pH 7.4, 300 mM NaCl, and 150 mM imidazole. The eluted fractions containing the purified protein were pooled and then subjected to overnight dialysis against 50 mM Tris–HCl pH 7.5, 50 mM NaCl, and 10% glycerol. The protein, if needed, was concentrated using Amicon Ultra115 centrifugal filter (UFC901008) using the protocol of the manufacturer.

Rotoli S.M., Jones J.L, & Caradonna S.J. (2018). Cysteine residues contribute to the dimerization and enzymatic activity of human nuclear dUTP nucleotidohydrolase (nDut). Protein Science : A Publication of the Protein Society, 27(10), 1797-1809.

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Molecular Cloning and Protein Purification

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Restriction enzymes and the Gibson Assembly kit were from New England BioLabs (Ipswich, MA, USA). Taq polymerase was from KAPA Biosystems (Wilmington, MA, USA). Phusion polymerase was from ThermoFisher Scientific (Waltham, MA, USA). Oligonucleotides were from Integrated DNA Technologies (Coralville, IA, USA). Bugbuster reagent and reduced NADH were from Merck Millipore (Billerica, MA, USA). Phenazine methosulfate (PMS) was from J. T. Baker Chemical Co. (Centre Valley, PA, USA). l-Arabinose and ampicillin were from Gold Biotechnology (St. Louis, MO, USA). Dithiothreitol (DTT) was from Melford Laboratories (Ipswich, UK). Acetone, butanone, acetoin, protease inhibitor cocktail and reduced NADPH were from Sigma Chemical Co. (St. Louis, MO, USA). 4-Nitroblue tetrazolium (NBT) chloride was from Boehringer Mannheim (Stuttgart, Germany). The QuikChange II Site-Directed Mutagenesis Kit, including Escherichia coli strain XL1-Blue, was from Agilent (Santa Clara, CA, USA). Talon metal affinity resin was from ClonTech (Mountain View, CA, USA).

Maddock D.J., Patrick W.M, & Gerth M.L. (2015). Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity. Protein Engineering, Design and Selection, 28(8), 251-258.

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Preparing Cell Extracts from C. thermocellum

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Escherichia coli cells were prepared as described in “Confirmation of PDC activity in C. thermocellum”. C. thermocellum cells were harvested by centrifugation when the absorbance at 600 nm reached a value of 0.6.
The cell pellet was resuspended in lysis buffer (1 × BugBuster reagent (EMD Millipore, Darmstadt, Germany) with 0.2 mM dithiothreitol). The cells were lysed with Ready-Lyse lysozyme (Epicentre, Madison, WI, USA), and DNase I (New England Biolabs, Ipswich, MA, USA) was added to reduce the viscosity. After incubation for 30 min at room temperature, the resulting solution was centrifuged at 10,000×g for 5 min. The supernatant was used as cell extract for enzyme assays or purification.

Tian L., Perot S.J., Hon S., Zhou J., Liang X., Bouvier J.T., Guss A.M., Olson D.G, & Lynd L.R. (2017). Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase. Microbial Cell Factories, 16, 171.

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Expression and Purification of Optimized Streptavidin

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To express SA variants, we transformed E. coli with the plasmid, pT7-7 STm and grown in M9 minimum medium supplemented with 100 μg ml⁻¹ ampicillin at 37 °C. Once the culture reached OD₆₀₀∼0.8, we induced the recombinant protein expression with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at 30 °C. We harvested and lysed cells with BugBuster reagent (EMD Millipore) supplemented with protease inhibitor cocktail III (EMD Millipore) and Lysonase Bioprocessing Reagent (EMD Millipore) for 30 min at room temperature. Insoluble fractions were removed by centrifugation at 10,000 g for 40 min. The soluble fraction of lysate was used for the affinity purification of optimized SA using T7-Tag Affinity Purification Kit (EMD Millipore) according to the manufacturer's instructions. We then buffer exchanged the purified protein and concentrated into the assay buffer. Wild-type SA was purchased from Sigma-Aldrich (St Louis, MO). Protein concentrations were determined using the 660 nm Protein Assay (Thermo Scientific) with BSA as a standard.

Matsumoto Y., Chen R., Anikeeva P, & Jasanoff A. (2015). Engineering intracellular biomineralization and biosensing by a magnetic protein. Nature Communications, 6, 8721.

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Purification of Recombinant KAI2 Proteins

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AtKAI2 and BtKAI2-coding sequences were cloned into pE-SUMO-Amp (LifeSensors) to generate N-terminal 6 × HIS-SUMO fusion proteins. All proteins were expressed in BL21 Rosetta DE3 pLysS cells (Merck-Millipore) and purified using IMAC as described in detail previously^{41 (link)}. In brief, cultures were grown in Luria-Bertani medium at 30 °C until the optical density reached 0.8–1, at which point the cultures were chilled to 16 °C and induced with 0.1 mM isopropyl β-d-1 thiogalactopyranoside and allowed to grow for a further 14–16 h. Pellets were lysed in 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM imidazole and 1 × BugBuster reagent (Merck-Millipore). Proteins were purified from lysates in batch mode using gravity columns containing TALON cobalt affinity resin (Takara). Columns were washed with 10 mM imidazole and eluted with 200 mM imidazole in 1-mL fractions. Proteins were concentrated and imidazole removed by ultrafiltration and buffer exchange into 20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol.

Sun Y.K., Yao J., Scaffidi A., Melville K.T., Davies S.F., Bond C.S., Smith S.M., Flematti G.R, & Waters M.T. (2020). Divergent receptor proteins confer responses to different karrikins in two ephemeral weeds. Nature Communications, 11, 1264.

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Recombinant Cel5A Protein Production

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A total of 150 mL of LB broth containing 30 μg/mL kanamycin was prepared and inoculated with transformed E. coli with pET41b-Cel5A then grown at 37°C for 3 h in a shaking incubator set at 180 rpm. After the optical density in 600 nm (OD₆₀₀) value of the culture reached around 1.0, Isopropyl β-D-1-thiogalactopyranoside (IPTG) 1 mM was added then shook overnight in 16°C. The cell pellet was collected after centrifugation at 10,000 g for 10 min and then lysed by Bugbuster reagent (Novagen’s pET Systems, Merck Millipore Corp., Germany). The cell lysate was harvested by centrifugation at 12,000 g for 20 min and the His-tagged Cel5A was purified using Ni-NTA His-Bind column (Novagen’s pET Systems, Merck Millipore Corp., Germany). A gradient of 5 to 250 mM imidazole in Tris-HCl buffer containing 500 mM NaCl was employed to purify the fusion protein. After elution of the target protein from the resin, the product was used for further characterization of its properties.

Park T., Seo S., Shin T., Cho B.W., Cho S., Kim B., Lee S., Ha J.K, & Seo J. (2017). Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers’ rumen. Asian-Australasian Journal of Animal Sciences, 31(4), 607-615.

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