Bugbuster protein extraction reagent

Manufactured by Merck Group

Sourced in United States, Germany

BugBuster Protein Extraction Reagent is a buffer solution designed to extract proteins from bacterial cells. It facilitates the lysis of cell membranes, allowing the release and recovery of target proteins for further analysis or purification.

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Lab products found in correlation

Benzonase nuclease, by Merck Group (8 mentions) Lysozyme, by Merck Group (3 mentions) Histrap hp column, by GE Healthcare (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions) Isopropyl β d thiogalactopyranoside iptg, by Fujifilm (2 mentions) Protein assay kit, by Bio-Rad (2 mentions) Rlysozme, by Merck Group (2 mentions) Omnicleave endonuclease, by Illumina (2 mentions) 13c6 lysine, by Merck Group (2 mentions) 13c6 arginine, by Merck Group (2 mentions) E coli bl21 de3 plyss cells, by Merck Group (1 mentions) E coli dh5 alpha, by New England Biolabs (1 mentions) Streptavidin dynabeads, by Thermo Fisher Scientific (1 mentions) Desalting column, by GE Healthcare (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail with edta, by Roche (1 mentions) Nylon membrane, by Merck Group (1 mentions) Ccd camera, by Intas (1 mentions) Ni nta agarose, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

BugBuster (71 citations) BugBuster reagent (20 citations) Protein extraction reagent (7 citations) BugBuster HT Protein Extraction Reagent (5 citations) Bugbuster 10× protein extraction reagent (2 citations)

64 protocols using bugbuster protein extraction reagent

Noncanonical Amino Acid Incorporation in E. coli

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The pBAD-cpTMV
vectors containing the relevant noncanonical amino acid amber codon
(or pBAD-sfGFP 150TAG for GFP controls) were cotransformed with pDule-3-nitroTyrosine
into DH10B E. coli cells and plated
on LB agar plates containing 50 μg/mL ampicillin and 12.5 μg/mL
tetracycline. The resulting colonies were grown overnight in 10 mL
of LB containing 50 μg/mL ampicillin and 12.5 μg/mL tetracycline
at 37 °C and then 25 μL of the overnight culture was added
to 5 mL of arabinose auto-induction media^{38 (link)} with modifications, as shown in Figure S1. The cultures were then shaken at 37 °C, 220 rpm. At the times
specified in Figure S1, 250 μL of
a 20 mM solution of the noncanonical amino acid (3NY) was added to
the growth medium to a final concentration of 1 mM. The culture was
incubated at 37 °C, 220 rpm for a total of 23 h. 500 μL
of culture from each expression was then centrifuged at 13,200 rpm
for 5 min. The supernatant was discarded, and the pellet was resuspended
in 250 μL of Bugbuster Protein Extraction Reagent (MilliporeSigma,
St. Louis, MO) and allowed to sit at room temperature for 20 min.
The solution was then centrifuged for 20 min at 13,200 rpm, the supernatant
was removed and retained, and the insoluble portion was resuspended
in lysis buffer. The expression level in the supernatant and resuspended
insoluble portions were analyzed using gel electrophoresis.

Bischoff A.J., Hamerlynck L.M., Li A.J., Roberts T.D., Ginsberg N.S, & Francis M.B. (2023). Protein-Based Model for Energy Transfer between Photosynthetic Light-Harvesting Complexes Is Constructed Using a Direct Protein–Protein Conjugation Strategy. Journal of the American Chemical Society, 145(29), 15827-15837.

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Borrelia burgdorferi Growth Conditions

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B. burgdorferi was grown at 35 °C until it reached the midlog phase. This culture was diluted to 10⁶ bacteria/mL and grown at 23 °C pH 7.6 until it reached a density of 1 × 10⁷ bacteria/mL. The culture was diluted to a density of 2.5 × 10⁵ bacteria/mL and separate cultures were incubated at 23 °C pH 7.6 and at 35 °C pH 6.8 until they reached a mid-exponential phase. Cultures at the same density were lysed using BugBuster protein extraction reagent (Millipore Sigma, St. Louis, MO, USA) and subsequently tested by Western blot and ELISA as previously described.

Cafiero T, & Toledo A. (2021). Borrelia burgdorferi Surface Exposed GroEL Is a Multifunctional Protein. Pathogens, 10(2), 226.

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Purification of E.coli Recombinant Proteins

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E.coli DH5 alpha were purchased from New England Biolabs (Ipswich, MA, USA). E.coli BL21(DE3) pLysS cells, BugBuster protein extraction reagent, and cOmplete™ EDTA-free protease inhibitor cocktail tablets were from Millipore-Sigma (St. Louis, MO, USA). Chelating and Q Sepharose were from Cytivia Inc. (Marlborough, MA, USA).

Doctor K.Z., Gilmour E., Recarte M., Beatty T.R., Shifa I., Stangel M., Schwisow J., Leary D.H, & Legler P.M. (2023). Automated SSHHPS Analysis Predicts a Potential Host Protein Target Common to Several Neuroinvasive (+)ssRNA Viruses. Viruses, 15(2), 542.

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Purification of rVacJ Protein

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Cell pellets containing rVacJ protein as inclusion bodies were re-suspended in BugBuster Protein Extraction Reagent (Millipore Sigma, Burlington, MA) in the presence of benzonase and r-Lysozyme. After centrifugation the pellet was re-suspended in BugBuster reagent and pelleted followed by four washes in dilute BugBuster reagent (1:10). The rVacJ containing pellet was dissolved in binding buffer (8 M urea, 0.1 M sodium phosphate buffer, 0.01 M Tris, pH 8.0) and loaded onto Ni NTA His-bind Superflow column (Novagen). The column was washed in binding buffer, then 8 M urea in 0.1 M sodium phosphate buffer, 0.01 M Tris, pH 6.5 before elution in 8 M urea in 0.1 M sodium phosphate buffer, 0.01 M Tris pH 4.5.

Hau S.J., Luan S.L., Loving C.L., Nicholson T.L., Wang J., Peters S.E., Seilly D., Weinert L.A., Langford P.R., Rycroft A.N., Wren B.W., Maskell D.J., Tucker A.W, & Brockmeier S.L. (2020). Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against Glaesserella parasuis in pigs. BMC Veterinary Research, 16, 167.

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Purification of MBP Fusion Proteins

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Expression of MBP fusion proteins was induced in BL21 cells with 1 mM IPTG at an optical density at 600 nm of 0.4–0.6 for 2 hr at 37°C. Cells were pelleted and lysed in 1x BugBuster Protein Extraction Reagent (Millipore Sigma 70584) for 20 min at 23°C. Cell lysates were centrifuged at 16,000xg at 4°C for 20 min to remove cellular debris.
MBP fusion proteins were batch purified from cleared lysates with amylose resin (New England Biolabs E8021S) in 1x amylose binding buffer (20 mM Tris-HCl pH7.4, 200 mM NaCl, 1 mM EDTA) overnight at 4°C. Resin was washed 10 times with 1x amylose binding buffer with 1% Triton X-100 and 0.5% deoxycholate. Purified MBP fusion proteins were batch eluted in 1x amylose elution buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM maltose).
Purified MBP fusion proteins were captured with biotin-peptide coated Streptavidin Dynabeads (Thermo Fisher Scientific 11047) overnight at 4°C in 1x PBS. The next day, Streptavidin Dynabeads were washed 10 times in 1x RIPA buffer, and captured proteins, along with input controls, were resolved by SDS-PAGE and visualized by Coomassie Blue staining.

Cho C., Wang Y., Smallwood P.M., Williams J, & Nathans J. (2019). Dlg1 activates beta-catenin signaling to regulate retinal angiogenesis and the blood-retina and blood-brain barriers. eLife, 8, e45542.

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Purification of Recombinant Laccase Mutants

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The 19 pECtLac mutants were separately cloned in E. coli strain BL21(DE3) for protein expression. The E. coli strain BL21(DE3) harboring plasmid with each CtLac V243 mutant was grown, and protein expression conducted as described above. After the protein induction, cells were harvested by centrifuge at 10,000×g for 15 min and washed twice with minimal salts basal medium for protein purification. Cell lysis was conducted using BugBuster™ protein extraction reagent (Sigma-Aldrich) according to the manufacturer’s protocol. The lysate was cleared by centrifugation and filtration using a 0.2-μm membrane filter (Advantec MFS Inc., Dublin, CA, USA) and used as a crude laccase enzyme source or further purified to homogeneity using HisTrap affinity chromatography. The lysate was applied in a His-Trap HP column (GE Healthcare, Chicago, IL, USA) and eluted with a linear gradient of imidazole from 0 to 250 mM buffered 20 mM Tris–HCl (pH 8.0). A fraction containing the laccase was desalted using a desalting column (GE Healthcare) and then concentrated using an Ultra Centrifugal Filter (Amicon, Miami, FL) to obtain concentrated high-purity protein for further experiments. The protein concentration was determined by a BCA protein assay kit (Pierce Biotechnology, Waltham, MA).

Yang Y., Ghatge S, & Hur H.G. (2022). Improvement of thermoalkaliphilic laccase (CtLac) by a directed evolution and application to lignin degradation. Applied Microbiology and Biotechnology, 107(1), 273-286.

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Leptospira Protein Extraction Protocol

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The L. interrogans serovars Lai, Canicola, Copenhageni, and the non-pathogenic serovar L. biflexa serovar Patoc were grown in liquid EMJH medium and harvested by centrifugation at 18,514 g for 10 mins. Cells were washed twice with 1X PBS pH 7.4 and pellets were resuspended in 5 mL/gram of BugBuster^® Protein Extraction Reagent (Sigma-Aldrich, USA) containing “Protease Inhibitor Cocktail with EDTA” (Roche, USA). Cell lysates were incubated on a rotating mixer for 15 minutes at room temperature. Insoluble cell debris was removed by centrifugation at 18,514 g for 20 minutes at 4°C. Supernatant were stored at -20°C until analysis.

Chaurasia R., Salovey A., Guo X., Desir G, & Vinetz J.M. (2022). Vaccination With Leptospira interrogans PF07598 Gene Family-Encoded Virulence Modifying Proteins Protects Mice From Severe Leptospirosis and Reduces Bacterial Load in the Liver and Kidney. Frontiers in Cellular and Infection Microbiology, 12, 926994.

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Purification of His-tagged hCD47 Proteins

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His‐hCD47 WT and His‐hCD47 Y288F were expressed in BL21 (DE3) cells and then purified by His‐NTA resin (GE Healthcare, Pittsburgh, PA).^[²³^] Briefly, pCold His‐CD47 WT and pCold hCD47 Y288F were transformed into BL21/DE3 bacteria. Transformants were screened and selected to inoculate 10 mL cultures of LB/ampicillin, which were grown overnight at 37 °C to stationary phase. 2 mL of precultured bacterial medium was then used to inoculate 50 mL of LB/ampicillin. The cultures were grown at 37 °C to an OD600 of ≈0.4–0.6 before the addition of 0.5 mm IPTG at 16 °C for 24 h. After that, cell pellets were collected by centrifuging at 5000 r.p.m. for 5 min at 4 °C, resuspended in 10 mL Bugbuster protein extraction reagent (Sigma‒Aldrich) with the addition of 20 µL protease cocktail inhibitor (Roche), and incubated at room temperature for 20 min before centrifuging at 10 000 r.p.m. for 10 min at 4 °C. Cleared lysates were then bound to Ni‐NTA His Bind Resin for 12 h with rolling at 4 °C. The beads were washed with extraction buffer for 5 min and rolled at 4 °C three times. Then, the beads were collected and eluted with extraction buffer (pH 7.5) plus 500 mm imidazole for 1 h with rolling at 4 °C. The purified proteins were then dialyzed at 20 mm Tris‐HCl pH 7.5, 50 mm NaCl, 10% glycerol, and 1 mm dithiothreitol at 4 °C overnight.

Du L., Su Z., Wang S., Meng Y., Xiao F., Xu D., Li X., Qian X., Lee S.B., Lee J., Lu Z, & Lyu J. (2023). EGFR‐Induced and c‐Src‐Mediated CD47 Phosphorylation Inhibits TRIM21‐Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion. Advanced Science, 10(27), 2206380.

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Purification of WT and mutant BspC V-domains

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Both the WT BspC V-domain and the gating loop mutant BspC V-domain proteins were purified in the same manner. WT and GLM V-domain coding sequence was cloned into the pTEV5 vector with an N-terminal His6-tag fusion and expressed in E. coli BL21; pLysS while shaking in LB at 37°C with the addition of 1 mM IPTG for 6 hours. Bacteria was pelleted and lysed with BugBuster Protein Extraction Reagent (Sigma). The lysate was centrifuged, and protein was purified from supernatant using a HIS-Select Nickel Affinity Gel Column (Sigma). The His6-tag was then cleaved using the TEV protease, and then the proteins were repurified with using the HIS-Select Nickel Affinity Gel Column.

Manzer H.S., Villarreal R.I, & Doran K.S. (2022). Targeting the BspC-vimentin interaction to develop anti-virulence therapies during Group B streptococcal meningitis. PLoS Pathogens, 18(3), e1010397.

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Preparation of E. coli Lysates

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The vh/vhh-phagemid-transformed HB2151 E. coli clones were grown in 10 mL of LB broth at 37 °C with shaking aeration (250 rpm) overnight. After centrifugation (10,000× g, 4 °C for 5 min), the bacteria in each pellet were resuspended in 1 mL of PBS and subjected to sonication for 90 s (30% power, 5 s pulse-on and 1 s pulse-off). The preparation was centrifuged (15,000× g, 4 °C for 5 min), and the supernatants (E. coli lysates) were collected. Alternatively, lysates of the E. coli were prepared by adding 500 µL of 1× BugBuster^® protein extraction reagent (Millipore, Merck KGaA, Darmstadt, Germany) to the bacterial pellet derived from 10 mL of log-phase-grown culture. After centrifugation (15,000× g, 4 °C for 5 min), the E. coli lysates (supernatants) were collected.

Kaewchim K., Glab-ampai K., Mahasongkram K., Saenlom T., Thepsawat W., Chulanetra M., Choowongkomon K., Sookrung N, & Chaicumpa W. (2023). Neutralizing and Enhancing Epitopes of the SARS-CoV-2 Receptor-Binding Domain (RBD) Identified by Nanobodies. Viruses, 15(6), 1252.

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