recombinant mutyh proteins e coli bl21 codonplus de3 rp competent cells  (Stratagene)

 
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    Structured Review

    Stratagene recombinant mutyh proteins e coli bl21 codonplus de3 rp competent cells
    Measurement of DNA glycosylase activity of <t>MUTYH</t> type 1 protein. ( A ) Purification of wild-type MUTYH type 1 protein resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue (CBB). The human MUTYH type 1 cDNA sequence was inserted into a pET25b(+) expression vector (Novagen, Darmstadt, Germany). E. coli <t>BL21-CodonPlus</t> (DE3)-RP competent cells (Stratagene, La Jolla, CA) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A 600 of 0.6. After incubation with 0.1 mM IPTG at 20°C for 12 h, MUTYH-His 6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech). The protein was then dialyzed against buffer containing 10 mM sodium phosphate (pH 7.6), 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA, 0.5 mM PMSF, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 50 μM chymostatin, and 10% glycerol. Lysates of E. coli culture without or with IPTG induction and purified MUTYH type 1 protein are shown. The arrow points to the MUTYH-His 6 protein band. ( B ) Western blot of purified wild-type MUTYH type 1 protein tagged with His 6 . Purified recombinant NEIL1-His 6 protein, which was prepared by using pET25b(+) vector (Novagen) and E. coli BL21-CodonPlus (DE3)-RP cells (Stratagene) previously ( Shinmura et al., 2004 ), was included as a negative control. Purified recombinant protein was mixed with an equal volume of 2x SDS sample buffer and boiled. A 2 ug protein was subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare Bio-Science Corp., Piscataway,NJ). The membrane was blocked with nonfat milk and incubated with an anti-MUTYH polyclonal antibody ( Ohtsubo et al., 2000 ). After washing, the membrane was incubated with an anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Bio-Science Corp.). The membrane was then washed, and immunoreactivity was visualized with an ECL Plus chemiluminescence system (GE Healthcare Bio-Science Corp.). MUTYH-His 6 protein is indicated by the arrow. ( C ) The DNA glycosylase activity of wild-type MUTYH type 1 protein on double-stranded DNA containing an A:8-hydroxyguanine (8-OHG). 30-mer oligonucleotides containing and not containing a single 8-OHG (5′-CTG GTG GCC TGA C[8-OHG or T]C ATT CCC CAA CTA GTG-3′) were chemically synthesized and purified by PAGE (Japan Bio Services, Saitama, Japan). Complementary oligonucleotides containing an adenine opposite the 8-OHG or T were 32 P-labeled at the 5′ terminus with a MEGALABEL kit (Takara, Osaka, Japan) and a [γ- 32 P]ATP (PerkinElmer, Tokyo, Japan), and then annealed to oligonucleotides containing a single 8-OHG or T. The reaction mixture containing 20 mM sodium phosphate (pH 7.6), 100 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 5 μM ZnCl 2 , 1.5% glycerol, 2.5 nM labeled oligonucleotide, 50 μg/ml BSA, and purified MUTYH protein was incubated at 37°C, and the mixture was treated with 0.1 M NaOH at 95°C for 4 min. After adding denaturing formamide dye to the mixture, it was heated at 95°C for 3 min, and subjected to 20% PAGE. A 32 P-labeled marker oligonucleotide was used as a size marker for the cleavage products. The radioactivity of intact and cleaved oligonucleotides was quantified by using an FLA-3000 fluoroimage analyzer (Fuji Film, Tokyo, Japan) and ImageGauge software (Fuji Film) ( Goto et al., 2009 ). The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. Heat-inactivation of the MUTYH protein was accomplished by heating the protein at 100°C for 5 min. 8G means 8-hydroxyguanine. ( D ) Time-course assay of cleavage of DNA containing an A:8-OHG by wild-type MUTYH type 1 protein. The MUTYH type 1 protein (260 fmole) was incubated at 37°C for 0 - 60 min with double-stranded oligonucleotide containing an A:8-OHG (50 fmole). The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision. The % incision values are shown as means ± standard errors of data from three independent experiments. ( E ) DNA glycosylase activities of wild-type MUTYH type 1 protein and their variant proteins on an A:8-OHG substrate. DNA cleavage activities of MUTYH type 1 proteins were measured at 37°C for 15 min. The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision, and the % incision of each variant-type MUTYH protein is shown relative to that of wild-type (WT) MUTYH protein, which has been set equal to 100. Values are means ± standard errors of data from three independent experiments. ND, not detected.
    Recombinant Mutyh Proteins E Coli Bl21 Codonplus De3 Rp Competent Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 78/100, based on 337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer"

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer

    Journal: Human Mutation

    doi: 10.1002/humu.21363

    Measurement of DNA glycosylase activity of MUTYH type 1 protein. ( A ) Purification of wild-type MUTYH type 1 protein resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue (CBB). The human MUTYH type 1 cDNA sequence was inserted into a pET25b(+) expression vector (Novagen, Darmstadt, Germany). E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene, La Jolla, CA) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A 600 of 0.6. After incubation with 0.1 mM IPTG at 20°C for 12 h, MUTYH-His 6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech). The protein was then dialyzed against buffer containing 10 mM sodium phosphate (pH 7.6), 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA, 0.5 mM PMSF, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 50 μM chymostatin, and 10% glycerol. Lysates of E. coli culture without or with IPTG induction and purified MUTYH type 1 protein are shown. The arrow points to the MUTYH-His 6 protein band. ( B ) Western blot of purified wild-type MUTYH type 1 protein tagged with His 6 . Purified recombinant NEIL1-His 6 protein, which was prepared by using pET25b(+) vector (Novagen) and E. coli BL21-CodonPlus (DE3)-RP cells (Stratagene) previously ( Shinmura et al., 2004 ), was included as a negative control. Purified recombinant protein was mixed with an equal volume of 2x SDS sample buffer and boiled. A 2 ug protein was subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare Bio-Science Corp., Piscataway,NJ). The membrane was blocked with nonfat milk and incubated with an anti-MUTYH polyclonal antibody ( Ohtsubo et al., 2000 ). After washing, the membrane was incubated with an anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Bio-Science Corp.). The membrane was then washed, and immunoreactivity was visualized with an ECL Plus chemiluminescence system (GE Healthcare Bio-Science Corp.). MUTYH-His 6 protein is indicated by the arrow. ( C ) The DNA glycosylase activity of wild-type MUTYH type 1 protein on double-stranded DNA containing an A:8-hydroxyguanine (8-OHG). 30-mer oligonucleotides containing and not containing a single 8-OHG (5′-CTG GTG GCC TGA C[8-OHG or T]C ATT CCC CAA CTA GTG-3′) were chemically synthesized and purified by PAGE (Japan Bio Services, Saitama, Japan). Complementary oligonucleotides containing an adenine opposite the 8-OHG or T were 32 P-labeled at the 5′ terminus with a MEGALABEL kit (Takara, Osaka, Japan) and a [γ- 32 P]ATP (PerkinElmer, Tokyo, Japan), and then annealed to oligonucleotides containing a single 8-OHG or T. The reaction mixture containing 20 mM sodium phosphate (pH 7.6), 100 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 5 μM ZnCl 2 , 1.5% glycerol, 2.5 nM labeled oligonucleotide, 50 μg/ml BSA, and purified MUTYH protein was incubated at 37°C, and the mixture was treated with 0.1 M NaOH at 95°C for 4 min. After adding denaturing formamide dye to the mixture, it was heated at 95°C for 3 min, and subjected to 20% PAGE. A 32 P-labeled marker oligonucleotide was used as a size marker for the cleavage products. The radioactivity of intact and cleaved oligonucleotides was quantified by using an FLA-3000 fluoroimage analyzer (Fuji Film, Tokyo, Japan) and ImageGauge software (Fuji Film) ( Goto et al., 2009 ). The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. Heat-inactivation of the MUTYH protein was accomplished by heating the protein at 100°C for 5 min. 8G means 8-hydroxyguanine. ( D ) Time-course assay of cleavage of DNA containing an A:8-OHG by wild-type MUTYH type 1 protein. The MUTYH type 1 protein (260 fmole) was incubated at 37°C for 0 - 60 min with double-stranded oligonucleotide containing an A:8-OHG (50 fmole). The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision. The % incision values are shown as means ± standard errors of data from three independent experiments. ( E ) DNA glycosylase activities of wild-type MUTYH type 1 protein and their variant proteins on an A:8-OHG substrate. DNA cleavage activities of MUTYH type 1 proteins were measured at 37°C for 15 min. The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision, and the % incision of each variant-type MUTYH protein is shown relative to that of wild-type (WT) MUTYH protein, which has been set equal to 100. Values are means ± standard errors of data from three independent experiments. ND, not detected.
    Figure Legend Snippet: Measurement of DNA glycosylase activity of MUTYH type 1 protein. ( A ) Purification of wild-type MUTYH type 1 protein resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue (CBB). The human MUTYH type 1 cDNA sequence was inserted into a pET25b(+) expression vector (Novagen, Darmstadt, Germany). E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene, La Jolla, CA) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A 600 of 0.6. After incubation with 0.1 mM IPTG at 20°C for 12 h, MUTYH-His 6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech). The protein was then dialyzed against buffer containing 10 mM sodium phosphate (pH 7.6), 50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA, 0.5 mM PMSF, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 50 μM chymostatin, and 10% glycerol. Lysates of E. coli culture without or with IPTG induction and purified MUTYH type 1 protein are shown. The arrow points to the MUTYH-His 6 protein band. ( B ) Western blot of purified wild-type MUTYH type 1 protein tagged with His 6 . Purified recombinant NEIL1-His 6 protein, which was prepared by using pET25b(+) vector (Novagen) and E. coli BL21-CodonPlus (DE3)-RP cells (Stratagene) previously ( Shinmura et al., 2004 ), was included as a negative control. Purified recombinant protein was mixed with an equal volume of 2x SDS sample buffer and boiled. A 2 ug protein was subjected to SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (GE Healthcare Bio-Science Corp., Piscataway,NJ). The membrane was blocked with nonfat milk and incubated with an anti-MUTYH polyclonal antibody ( Ohtsubo et al., 2000 ). After washing, the membrane was incubated with an anti-rabbit HRP-conjugated secondary antibody (GE Healthcare Bio-Science Corp.). The membrane was then washed, and immunoreactivity was visualized with an ECL Plus chemiluminescence system (GE Healthcare Bio-Science Corp.). MUTYH-His 6 protein is indicated by the arrow. ( C ) The DNA glycosylase activity of wild-type MUTYH type 1 protein on double-stranded DNA containing an A:8-hydroxyguanine (8-OHG). 30-mer oligonucleotides containing and not containing a single 8-OHG (5′-CTG GTG GCC TGA C[8-OHG or T]C ATT CCC CAA CTA GTG-3′) were chemically synthesized and purified by PAGE (Japan Bio Services, Saitama, Japan). Complementary oligonucleotides containing an adenine opposite the 8-OHG or T were 32 P-labeled at the 5′ terminus with a MEGALABEL kit (Takara, Osaka, Japan) and a [γ- 32 P]ATP (PerkinElmer, Tokyo, Japan), and then annealed to oligonucleotides containing a single 8-OHG or T. The reaction mixture containing 20 mM sodium phosphate (pH 7.6), 100 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 5 μM ZnCl 2 , 1.5% glycerol, 2.5 nM labeled oligonucleotide, 50 μg/ml BSA, and purified MUTYH protein was incubated at 37°C, and the mixture was treated with 0.1 M NaOH at 95°C for 4 min. After adding denaturing formamide dye to the mixture, it was heated at 95°C for 3 min, and subjected to 20% PAGE. A 32 P-labeled marker oligonucleotide was used as a size marker for the cleavage products. The radioactivity of intact and cleaved oligonucleotides was quantified by using an FLA-3000 fluoroimage analyzer (Fuji Film, Tokyo, Japan) and ImageGauge software (Fuji Film) ( Goto et al., 2009 ). The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. Heat-inactivation of the MUTYH protein was accomplished by heating the protein at 100°C for 5 min. 8G means 8-hydroxyguanine. ( D ) Time-course assay of cleavage of DNA containing an A:8-OHG by wild-type MUTYH type 1 protein. The MUTYH type 1 protein (260 fmole) was incubated at 37°C for 0 - 60 min with double-stranded oligonucleotide containing an A:8-OHG (50 fmole). The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision. The % incision values are shown as means ± standard errors of data from three independent experiments. ( E ) DNA glycosylase activities of wild-type MUTYH type 1 protein and their variant proteins on an A:8-OHG substrate. DNA cleavage activities of MUTYH type 1 proteins were measured at 37°C for 15 min. The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision, and the % incision of each variant-type MUTYH protein is shown relative to that of wild-type (WT) MUTYH protein, which has been set equal to 100. Values are means ± standard errors of data from three independent experiments. ND, not detected.

    Techniques Used: Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Expressing, Plasmid Preparation, Transformation Assay, Cell Culture, Incubation, Western Blot, Recombinant, Negative Control, SDS Page, CTG Assay, Countercurrent Chromatography, Cellular Antioxidant Activity Assay, Synthesized, Labeling, Marker, Radioactivity, Software, Variant Assay

    Measurement of the adenine DNA glycosylase activity of wild-type MUTYH type 2 protein. ( A ) Purification of wild-type MUTYH type 2 protein resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue. Lysates of E. coli culture without or with IPTG induction and purified MUTYH type 2 protein are shown. The arrow points to the MUTYH-His 6 protein band. ( B ) Western blot of purified wild-type MUTYH type 2 protein tagged with His 6 . MUTYH-His 6 protein is indicated by the arrow. Purified recombinant NEIL1 (MIM# 608844)-His 6 protein, which was prepared by using pET25b(+) vector (Novagen) and E. coli BL21-CodonPlus (DE3)-RP cells (Stratagene) previously ( Shinmura et al., 2004 ), was included as a negative control. ( C ) The DNA glycosylase activity of MUTYH type 2 protein on double-stranded DNA containing an A:8-hydroxyguanine (8-OHG). The MUTYH type 2 protein and a 32 P-labeled double-stranded oligonucleotides containing or not containing a single 8-OHG mispair were incubated and subjected to 20% PAGE. The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. Heat-inactivation of the MUTYH protein was accomplished by heating the protein at 100°C for 5 min. 8G means 8-hydroxyguanine. ( D ) Protein concentration dependency of cleavage of DNA containing an A: 8-OHG by MUTYH type 2 protein. The MUTYH protein (2, 8, and 20 ng) was incubated at 37°C for 15 min with a 30-mer oligonucleotide containing an A: 8-OHG or A:T (50 fmole). The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision. ( E ) Time-course assay of cleavage of DNA containing an A: 8-OHG by MUTYH type 2 protein. The 8 ng amount of MUTYH type 2 protein was incubated at 37°C for 0-60 min with double-stranded oligonucleotide containing an A: 8-OHG (50 fmole). The % incision valves are means ± standard errors of data from three independent experiments.
    Figure Legend Snippet: Measurement of the adenine DNA glycosylase activity of wild-type MUTYH type 2 protein. ( A ) Purification of wild-type MUTYH type 2 protein resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie Brilliant Blue. Lysates of E. coli culture without or with IPTG induction and purified MUTYH type 2 protein are shown. The arrow points to the MUTYH-His 6 protein band. ( B ) Western blot of purified wild-type MUTYH type 2 protein tagged with His 6 . MUTYH-His 6 protein is indicated by the arrow. Purified recombinant NEIL1 (MIM# 608844)-His 6 protein, which was prepared by using pET25b(+) vector (Novagen) and E. coli BL21-CodonPlus (DE3)-RP cells (Stratagene) previously ( Shinmura et al., 2004 ), was included as a negative control. ( C ) The DNA glycosylase activity of MUTYH type 2 protein on double-stranded DNA containing an A:8-hydroxyguanine (8-OHG). The MUTYH type 2 protein and a 32 P-labeled double-stranded oligonucleotides containing or not containing a single 8-OHG mispair were incubated and subjected to 20% PAGE. The intact 30-mer oligonucleotides and cleavage products are indicated by the arrows. Heat-inactivation of the MUTYH protein was accomplished by heating the protein at 100°C for 5 min. 8G means 8-hydroxyguanine. ( D ) Protein concentration dependency of cleavage of DNA containing an A: 8-OHG by MUTYH type 2 protein. The MUTYH protein (2, 8, and 20 ng) was incubated at 37°C for 15 min with a 30-mer oligonucleotide containing an A: 8-OHG or A:T (50 fmole). The amount of cleavage products as a proportion of total oligonucleotides was calculated as % incision. ( E ) Time-course assay of cleavage of DNA containing an A: 8-OHG by MUTYH type 2 protein. The 8 ng amount of MUTYH type 2 protein was incubated at 37°C for 0-60 min with double-stranded oligonucleotide containing an A: 8-OHG (50 fmole). The % incision valves are means ± standard errors of data from three independent experiments.

    Techniques Used: Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Recombinant, Plasmid Preparation, Negative Control, Labeling, Incubation, Protein Concentration

    2) Product Images from "Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals 1"

    Article Title: Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals 1

    Journal: Plant Physiology

    doi: 10.1104/pp.102.018572

    Identification by GC-MS of the acetate esters product formed in vitro by the product of RhAAT1 . Bacterial lysates overexpressing RhAAT 1 were incubated with geraniol and acetyl-CoA in assay buffer as described in “Materials and Methods.” The identification of the geranyl acetate product was done by matching with the retention time and the mass spectrum of authentic geranyl acetate, and by comparison with the computerized Wiley library. The Kovac index of geranyl acetate also corresponded with that of authentic standard. A, Lysates (derived from cells overexpressing RhAAT1) + geraniol + acetyl-CoA. B, Reaction buffer + geraniol + acetyl-CoA. C, Lysates derived from cells overexpressing RhAAT1 + acetyl-CoA. D, Lysates derived from cells overexpressing RhAAT1 + geraniol (no acetyl-CoA). E, Control lysates (derived from control E. coli BL21 [DE3] p LysS cells, not overexpressing RhAAT1) + geraniol + acetyl-CoA.
    Figure Legend Snippet: Identification by GC-MS of the acetate esters product formed in vitro by the product of RhAAT1 . Bacterial lysates overexpressing RhAAT 1 were incubated with geraniol and acetyl-CoA in assay buffer as described in “Materials and Methods.” The identification of the geranyl acetate product was done by matching with the retention time and the mass spectrum of authentic geranyl acetate, and by comparison with the computerized Wiley library. The Kovac index of geranyl acetate also corresponded with that of authentic standard. A, Lysates (derived from cells overexpressing RhAAT1) + geraniol + acetyl-CoA. B, Reaction buffer + geraniol + acetyl-CoA. C, Lysates derived from cells overexpressing RhAAT1 + acetyl-CoA. D, Lysates derived from cells overexpressing RhAAT1 + geraniol (no acetyl-CoA). E, Control lysates (derived from control E. coli BL21 [DE3] p LysS cells, not overexpressing RhAAT1) + geraniol + acetyl-CoA.

    Techniques Used: Gas Chromatography-Mass Spectrometry, In Vitro, Incubation, Derivative Assay

    3) Product Images from "Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance"

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance

    Journal: The ISME Journal

    doi: 10.1038/ismej.2013.123

    Effect of salt stress on the growth rate of E. coli BL21 DE3 harboring constructs expressing the lysdh–aasadh operon components. ( a ) SDS-PAGE of proteins extracted from bacterial cells grown for 8 h in non-induced and IPTG-induced cultures.
    Figure Legend Snippet: Effect of salt stress on the growth rate of E. coli BL21 DE3 harboring constructs expressing the lysdh–aasadh operon components. ( a ) SDS-PAGE of proteins extracted from bacterial cells grown for 8 h in non-induced and IPTG-induced cultures.

    Techniques Used: Construct, Expressing, SDS Page

    4) Product Images from "The funnel approach to the pre-crystallization production of membrane proteins"

    Article Title: The funnel approach to the pre-crystallization production of membrane proteins

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2007.12.059

    Expression in whole cell lysates: (A) Growth in an automated plate reader of E. coli BL21 (DE3) Gold expressing 48 different pBAD constructs (left panel) and 48 different pET constructs (Right panel). Arrows indicate time of induction. (B) Dot blot analysis
    Figure Legend Snippet: Expression in whole cell lysates: (A) Growth in an automated plate reader of E. coli BL21 (DE3) Gold expressing 48 different pBAD constructs (left panel) and 48 different pET constructs (Right panel). Arrows indicate time of induction. (B) Dot blot analysis

    Techniques Used: Expressing, Construct, Positron Emission Tomography, Dot Blot

    5) Product Images from "Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine"

    Article Title: Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2013.10.011

    A biochemical approach to dissect portal function. (a) The plasmids containing g20 variants (WT or mutants) were transformed into E. coli BL21 (DE3) RIPL strain for IPTG induced expression of the respective gp20 protein (green). (b) The E. coli cells
    Figure Legend Snippet: A biochemical approach to dissect portal function. (a) The plasmids containing g20 variants (WT or mutants) were transformed into E. coli BL21 (DE3) RIPL strain for IPTG induced expression of the respective gp20 protein (green). (b) The E. coli cells

    Techniques Used: Transformation Assay, Expressing

    6) Product Images from "The funnel approach to the pre-crystallization production of membrane proteins"

    Article Title: The funnel approach to the pre-crystallization production of membrane proteins

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2007.12.059

    Expression in whole cell lysates: (A) Growth in an automated plate reader of E. coli BL21 (DE3) Gold expressing 48 different pBAD constructs (left panel) and 48 different pET constructs (Right panel). Arrows indicate time of induction. (B) Dot blot analysis
    Figure Legend Snippet: Expression in whole cell lysates: (A) Growth in an automated plate reader of E. coli BL21 (DE3) Gold expressing 48 different pBAD constructs (left panel) and 48 different pET constructs (Right panel). Arrows indicate time of induction. (B) Dot blot analysis

    Techniques Used: Expressing, Construct, Positron Emission Tomography, Dot Blot

    7) Product Images from "The Alternative Sigma Factor ?B of Bacillus cereus: Response to Stress and Role in Heat Adaptation"

    Article Title: The Alternative Sigma Factor ?B of Bacillus cereus: Response to Stress and Role in Heat Adaptation

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.2.316-325.2004

    Overproduction and purification of σ B . (Left panel) SDS-PAGE of protein extracts from E. coli BL21-Codonplus-(DE3)-RIL carrying either pET28-b or pMT01. (Right panel) Immunodetection of σ B with anti-σ B antiserum. Ten micrograms of protein was loaded for each sample. Lane 1, crude protein extract from E. coli carrying pET28-b; lane 2, crude protein extract from E. coli carrying pMT01; lane 3, crude protein extract from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 4, inclusion bodies isolated from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 5, representative fraction of purified σ B after elution from an Ni 2+ affinity column. The arrow indicates the position of the overproduced σ B protein.
    Figure Legend Snippet: Overproduction and purification of σ B . (Left panel) SDS-PAGE of protein extracts from E. coli BL21-Codonplus-(DE3)-RIL carrying either pET28-b or pMT01. (Right panel) Immunodetection of σ B with anti-σ B antiserum. Ten micrograms of protein was loaded for each sample. Lane 1, crude protein extract from E. coli carrying pET28-b; lane 2, crude protein extract from E. coli carrying pMT01; lane 3, crude protein extract from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 4, inclusion bodies isolated from E. coli carrying pMT01 after induction with 1 mM IPTG for 2 h; lane 5, representative fraction of purified σ B after elution from an Ni 2+ affinity column. The arrow indicates the position of the overproduced σ B protein.

    Techniques Used: Purification, SDS Page, Immunodetection, Isolation, Affinity Column

    8) Product Images from "Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine"

    Article Title: Structure-Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2013.10.011

    A biochemical approach to dissect portal function. (a) The plasmids containing g20 variants (WT or mutants) were transformed into E. coli BL21 (DE3) RIPL strain for IPTG induced expression of the respective gp20 protein (green). (b) The E. coli cells
    Figure Legend Snippet: A biochemical approach to dissect portal function. (a) The plasmids containing g20 variants (WT or mutants) were transformed into E. coli BL21 (DE3) RIPL strain for IPTG induced expression of the respective gp20 protein (green). (b) The E. coli cells

    Techniques Used: Transformation Assay, Expressing

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    Centrifugation:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
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    Article Snippet: Protein was expressed in Escherichia coli BL21(DE3)-Gold cells (Stratagene) with the RIL-Codon plus plasmid. .. The lysate was clarified by centrifugation twice at 50,000 g for 30 min. Hexahistidine–SUMO fusion proteins were isolated on a nickel-charged chelating column (GE Healthcare).

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Article Title: New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis
    Article Snippet: GST, GST‐MoPdeH, GST‐MoPdeHEAL(AAA) , GST‐MoPdeHΔ HD , GST‐MoPdeHΔ HD+L1L2 , GST‐MoPdeHΔ HD+L1 , GST‐MoPdeHΔ HD+L2 , GST‐MoPdeHHD , GST‐MoPdeLL1L2 , GST‐MoPdeLL1 , GST‐MoPdeLL2 and GST‐MoPdeL were expressed in Escherichia coli BL21‐CodonPlus (DE3) cells (Stratagene, Cedar Creek, TX, USA). .. Samples were induced with 0.1 m m IPTG (isopropyl‐β‐ d ‐1‐thiogalactopyranoside) at 18 °C for 4 h. After centrifugation at 2500 g for 10 min, the precipitated cells were resuspended in lysis buffer [1 × phosphate‐buffered saline (PBS), 10% glycerol].

    Amplification:

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: The amplified fragments were cloned into pET28a (Novagen, Darmstadt, DE) that had been double-digested with Nco I and Blp I. .. The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA).

    Synthesized:

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA). .. RNA and protein were extracted using the NucleoSpin RNA/Protein kit (Macherey–Nagel, Duren, DE). complementary DNA was synthesized and RT-PCR was performed to confirm the presence of lysdh and aasadh transcripts.

    Construct:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
    Article Snippet: Protein expression and purification We generated a hexahistidine–SUMO-tagged construct (pXC1010) of full length NgTet1 (321 amino acids; XP_002667965.1). .. The protein was expressed in E. coli BL21 (DE3)-Gold cells with the RIL-Codon plus plasmid (Stratagene).

    Article Title: The funnel approach to the pre-crystallization production of membrane proteins
    Article Snippet: Post induction, the pBAD constructs generally continued to grow normally, while the growth of many of the pET constructs stopped, with some cultures lysing. .. The initial expression studies for all clones utilized E. coli strain BL21 (DE3) GOLD™ (Stratagene).

    Article Title: The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix
    Article Snippet: We generated a hexahistidine–SUMO-tagged construct containing mouse UHRF1 residues 419−628 (pXC666). .. Protein was expressed in Escherichia coli BL21(DE3)-Gold cells (Stratagene) with the RIL-Codon plus plasmid.

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: .. The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA). ..

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: .. For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Incubation:

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. After incubation with 0.1 mM IPTG at 15°C for 12 h, MUTYH-His6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech).

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. The beads were then washed thrice, then suspended in glutathione elution buffer (50 mM Tris-HCl [pH 9.0], 500 mM NaCl, 50 mM glutathione, 1 mM DTT, 0.1 % Triton X-100) and incubated overnight at 4°C.

    Article Title: New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis
    Article Snippet: GST, GST‐MoPdeH, GST‐MoPdeHEAL(AAA) , GST‐MoPdeHΔ HD , GST‐MoPdeHΔ HD+L1L2 , GST‐MoPdeHΔ HD+L1 , GST‐MoPdeHΔ HD+L2 , GST‐MoPdeHHD , GST‐MoPdeLL1L2 , GST‐MoPdeLL1 , GST‐MoPdeLL2 and GST‐MoPdeL were expressed in Escherichia coli BL21‐CodonPlus (DE3) cells (Stratagene, Cedar Creek, TX, USA). .. The target proteins were collected by sonication and further purified with glutathione sepharose beads incubated at 4 °C for 3 h. After centrifugation at 500 g for 5 min, glutathione sepharose beads were washed with washing buffer [20 m m Tris (pH 7.5), 0.25 m m NaCl, 2 m m EDTA, 2 m m ethyleneglycol‐bis(β‐aminoethylether)‐ N , N '‐tetraacetic acid (EGTA)], eluted with washing buffer containing reduced glutathione (pH 8.0) (the elution contained 5% glycerol), concentrated at 4 °C and stored at −70 °C.

    Activity Assay:

    Article Title: New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis
    Article Snippet: Paragraph title: Detection of protein PDEase activity in vitro ... GST, GST‐MoPdeH, GST‐MoPdeHEAL(AAA) , GST‐MoPdeHΔ HD , GST‐MoPdeHΔ HD+L1L2 , GST‐MoPdeHΔ HD+L1 , GST‐MoPdeHΔ HD+L2 , GST‐MoPdeHHD , GST‐MoPdeLL1L2 , GST‐MoPdeLL1 , GST‐MoPdeLL2 and GST‐MoPdeL were expressed in Escherichia coli BL21‐CodonPlus (DE3) cells (Stratagene, Cedar Creek, TX, USA).

    Expressing:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
    Article Snippet: Paragraph title: Protein expression and purification ... The protein was expressed in E. coli BL21 (DE3)-Gold cells with the RIL-Codon plus plasmid (Stratagene).

    Article Title: Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1
    Article Snippet: The plasmid was transformed into the E. coli strain BL21 (DE3) RIL (Stratagene). .. The cells were cultured at 37 °C until the OD600 reached 0.8 and then the media was cooled to 20 °C and 0.2 mM IPTG was added to induce protein expression overnight.

    Article Title: The funnel approach to the pre-crystallization production of membrane proteins
    Article Snippet: .. The initial expression studies for all clones utilized E. coli strain BL21 (DE3) GOLD™ (Stratagene). .. Expression was tested at three different temperatures (37°, 28°, 23°), along with variations in duration of induction times and inducer concentrations (see Materials and Methods for details).

    Article Title: The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix
    Article Snippet: Paragraph title: Expression and purification ... Protein was expressed in Escherichia coli BL21(DE3)-Gold cells (Stratagene) with the RIL-Codon plus plasmid.

    Article Title: The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range
    Article Snippet: .. Protein expression and purification The proteins were expressed in the E. coli strain BL21 (DE3) RIL (Stratagene, La Jolla, USA) and purified slightly modified to remove the precursor tag according to standard protocols and stored in 40 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2 (standard buffer) at −80 °C until usage. .. For the NMR samples, minimal media containing 95% D2 O and supplemented with U-13 C glucose (Cambridge Isotope Laboratories, Tewksbury, USA) and 15 NH4 Cl (Cortecnet, Paris, France) were used for growing the cells and expressing the protein.

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: Paragraph title: Construction of vectors for LYSDH, AASADH and LYSDH/AASADH and expression in E. coli BL21 DE3 ... The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA).

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Article Title: Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals 1
    Article Snippet: Paragraph title: Expression of RhAAT1 in Escherichia coli ... Recombinant E. coli BL21 (DE3) Gold (Stratagene, La Jolla, CA) bacteria were plated in Luria-Bertani broth (LB)-agar containing 50 μg mL−1 ampicillin and 34 μg mL−1 chroramphenicol.

    Modification:

    Article Title: The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range
    Article Snippet: .. Protein expression and purification The proteins were expressed in the E. coli strain BL21 (DE3) RIL (Stratagene, La Jolla, USA) and purified slightly modified to remove the precursor tag according to standard protocols and stored in 40 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2 (standard buffer) at −80 °C until usage. .. For the NMR samples, minimal media containing 95% D2 O and supplemented with U-13 C glucose (Cambridge Isotope Laboratories, Tewksbury, USA) and 15 NH4 Cl (Cortecnet, Paris, France) were used for growing the cells and expressing the protein.

    Transformation Assay:

    Article Title: Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1
    Article Snippet: .. The plasmid was transformed into the E. coli strain BL21 (DE3) RIL (Stratagene). .. The cells were cultured at 37 °C until the OD600 reached 0.8 and then the media was cooled to 20 °C and 0.2 mM IPTG was added to induce protein expression overnight.

    Article Title: The funnel approach to the pre-crystallization production of membrane proteins
    Article Snippet: Standard transformation protocols were used, and transformed cells were plated onto an LB agar tray divided to a grid of 48 squares (Q-trays, Genetix USA). .. The initial expression studies for all clones utilized E. coli strain BL21 (DE3) GOLD™ (Stratagene).

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: .. Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. After incubation with 0.1 mM IPTG at 15°C for 12 h, MUTYH-His6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech).

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: .. Isotopically labeled 15 N-Aβ42 was prepared by growing transformed E. coli BL21 Gold (DE3) strain (Stratagene) in 1-liter flasks at 37°C with 500 ml of minimal M9 batch medium. ..

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: .. The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA). ..

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: .. For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Conjugation Assay:

    Article Title: The Alternative Sigma Factor ?B of Bacillus cereus: Response to Stress and Role in Heat Adaptation
    Article Snippet: E. coli BL21-Codonplus-(DE3)-RIL (Stratagene, La Jolla, Calif.) was used as the host for SigB overproduction. .. E. coli HB101/pRK24 ( ) was used as the donor host in conjugation experiments.

    Protease Inhibitor:

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Cell Culture:

    Article Title: Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1
    Article Snippet: The plasmid was transformed into the E. coli strain BL21 (DE3) RIL (Stratagene). .. The cells were cultured at 37 °C until the OD600 reached 0.8 and then the media was cooled to 20 °C and 0.2 mM IPTG was added to induce protein expression overnight.

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: .. Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. After incubation with 0.1 mM IPTG at 15°C for 12 h, MUTYH-His6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech).

    Article Title: The Alternative Sigma Factor ?B of Bacillus cereus: Response to Stress and Role in Heat Adaptation
    Article Snippet: All Escherichia coli strains were cultured in Luria broth at 37°C ( ). .. E. coli BL21-Codonplus-(DE3)-RIL (Stratagene, La Jolla, Calif.) was used as the host for SigB overproduction.

    Generated:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
    Article Snippet: Protein expression and purification We generated a hexahistidine–SUMO-tagged construct (pXC1010) of full length NgTet1 (321 amino acids; XP_002667965.1). .. The protein was expressed in E. coli BL21 (DE3)-Gold cells with the RIL-Codon plus plasmid (Stratagene).

    Article Title: The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix
    Article Snippet: We generated a hexahistidine–SUMO-tagged construct containing mouse UHRF1 residues 419−628 (pXC666). .. Protein was expressed in Escherichia coli BL21(DE3)-Gold cells (Stratagene) with the RIL-Codon plus plasmid.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA). .. RNA and protein were extracted using the NucleoSpin RNA/Protein kit (Macherey–Nagel, Duren, DE). complementary DNA was synthesized and RT-PCR was performed to confirm the presence of lysdh and aasadh transcripts.

    Sonication:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
    Article Snippet: The protein was expressed in E. coli BL21 (DE3)-Gold cells with the RIL-Codon plus plasmid (Stratagene). .. Cells were re-suspended with 4 volumes of 500 mM NaCl, 20 mM sodium phosphate, pH 7.4, 20 mM imidazole, 1 mM dithiothreitol (DTT) and 0.25 mM phenylmethyl-sulphonyl fluoride and sonicated for 5 min (1s on and 2s off).

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: Preparation of Aβ peptides The recombinant Aβ (M1-42) peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA), here called Aβ42, was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene) and purified as described previously with slight modifications ( ). .. Briefly, the purification procedure involved sonication of E. coli cells, dissolution of inclusion bodies in 8 M urea, ion exchange in batch mode on diethylaminoethyl cellulose resin, and lyophilization.

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: Preparation of Aβ42 for chemical kinetics experiments The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Article Title: New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis
    Article Snippet: GST, GST‐MoPdeH, GST‐MoPdeHEAL(AAA) , GST‐MoPdeHΔ HD , GST‐MoPdeHΔ HD+L1L2 , GST‐MoPdeHΔ HD+L1 , GST‐MoPdeHΔ HD+L2 , GST‐MoPdeHHD , GST‐MoPdeLL1L2 , GST‐MoPdeLL1 , GST‐MoPdeLL2 and GST‐MoPdeL were expressed in Escherichia coli BL21‐CodonPlus (DE3) cells (Stratagene, Cedar Creek, TX, USA). .. The target proteins were collected by sonication and further purified with glutathione sepharose beads incubated at 4 °C for 3 h. After centrifugation at 500 g for 5 min, glutathione sepharose beads were washed with washing buffer [20 m m Tris (pH 7.5), 0.25 m m NaCl, 2 m m EDTA, 2 m m ethyleneglycol‐bis(β‐aminoethylether)‐ N , N '‐tetraacetic acid (EGTA)], eluted with washing buffer containing reduced glutathione (pH 8.0) (the elution contained 5% glycerol), concentrated at 4 °C and stored at −70 °C.

    Recombinant:

    Article Title: Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1
    Article Snippet: The plasmid was transformed into the E. coli strain BL21 (DE3) RIL (Stratagene). .. The recombinant expressed protein was first purified using a HisTrap FF column (GE Healthcare).

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: .. The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: .. Preparation of Aβ peptides The recombinant Aβ (M1-42) peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA), here called Aβ42, was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene) and purified as described previously with slight modifications ( ). .. Briefly, the purification procedure involved sonication of E. coli cells, dissolution of inclusion bodies in 8 M urea, ion exchange in batch mode on diethylaminoethyl cellulose resin, and lyophilization.

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: .. Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. After incubation with 0.1 mM IPTG at 15°C for 12 h, MUTYH-His6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech).

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: The fractions containing the recombinant protein were combined, frozen using liquid nitrogen, and lyophilized again. .. Isotopically labeled 15 N-Aβ42 was prepared by growing transformed E. coli BL21 Gold (DE3) strain (Stratagene) in 1-liter flasks at 37°C with 500 ml of minimal M9 batch medium.

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA). .. To induce recombinant protein expression the four clones were grown in LB medium containing 50 μ M isopropyl β- D -1-thiogalactopyranoside (IPTG) (inducer) or 5 m M D -glucose (repressor).

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: .. For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: .. Preparation of Aβ42 for chemical kinetics experiments The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Article Title: Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals 1
    Article Snippet: .. Recombinant E. coli BL21 (DE3) Gold (Stratagene, La Jolla, CA) bacteria were plated in Luria-Bertani broth (LB)-agar containing 50 μg mL−1 ampicillin and 34 μg mL−1 chroramphenicol. ..

    Nucleic Acid Electrophoresis:

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The lyophilized fractions were further purified using a Superdex 75 26/60 column (GE healthcare, IL, USA), and the eluates were analysed using SDS-polyacrylamide gel electrophoresis to detect the presence of the Aβ42 peptide.

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: Preparation of Aβ peptides The recombinant Aβ (M1-42) peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA), here called Aβ42, was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene) and purified as described previously with slight modifications ( ). .. The lyophilized fractions were further purified using a Superdex 75 HR 26/60 column (GE Healthcare), and eluates were analyzed using SDS–polyacrylamide gel electrophoresis for the presence of the desired protein product.

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: Preparation of Aβ42 for chemical kinetics experiments The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The lyophilized fractions were further purified using a Superdex 75 26/60 column (GE healthcare, IL, USA), and the eluates were analysed using SDS-polyacrylamide gel electrophoresis to detect the presence of the Aβ42 peptide.

    Ion Exchange Chromatography:

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. Ion exchange chromatography was then performed on a diethyl-aminoethyl cellulose resin, and the protein collected was lyophilized.

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: Preparation of Aβ42 for chemical kinetics experiments The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. Ion exchange chromatography was then performed on a diethyl-aminoethyl cellulose resin, and the protein collected was lyophilized.

    Mutagenesis:

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: .. For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Isolation:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
    Article Snippet: The protein was expressed in E. coli BL21 (DE3)-Gold cells with the RIL-Codon plus plasmid (Stratagene). .. The lysate was clarified by centrifugation at 38,000 g for 60 min. Hexahistidine fusion protein was isolated on a nickel-charged chelating column (GE Healthcare).

    Article Title: The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix
    Article Snippet: Protein was expressed in Escherichia coli BL21(DE3)-Gold cells (Stratagene) with the RIL-Codon plus plasmid. .. The lysate was clarified by centrifugation twice at 50,000 g for 30 min. Hexahistidine–SUMO fusion proteins were isolated on a nickel-charged chelating column (GE Healthcare).

    Labeling:

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: .. Isotopically labeled 15 N-Aβ42 was prepared by growing transformed E. coli BL21 Gold (DE3) strain (Stratagene) in 1-liter flasks at 37°C with 500 ml of minimal M9 batch medium. ..

    Purification:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
    Article Snippet: Paragraph title: Protein expression and purification ... The protein was expressed in E. coli BL21 (DE3)-Gold cells with the RIL-Codon plus plasmid (Stratagene).

    Article Title: Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1
    Article Snippet: The plasmid was transformed into the E. coli strain BL21 (DE3) RIL (Stratagene). .. The recombinant expressed protein was first purified using a HisTrap FF column (GE Healthcare).

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: .. The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: .. Preparation of Aβ peptides The recombinant Aβ (M1-42) peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA), here called Aβ42, was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene) and purified as described previously with slight modifications ( ). .. Briefly, the purification procedure involved sonication of E. coli cells, dissolution of inclusion bodies in 8 M urea, ion exchange in batch mode on diethylaminoethyl cellulose resin, and lyophilization.

    Article Title: The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix
    Article Snippet: Paragraph title: Expression and purification ... Protein was expressed in Escherichia coli BL21(DE3)-Gold cells (Stratagene) with the RIL-Codon plus plasmid.

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. After incubation with 0.1 mM IPTG at 15°C for 12 h, MUTYH-His6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech).

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: The lyophilized fractions were further purified using a Superdex 75 HR 26/60 column (GE Healthcare), and eluates were analyzed using SDS–polyacrylamide gel electrophoresis for the presence of the desired protein product. .. Isotopically labeled 15 N-Aβ42 was prepared by growing transformed E. coli BL21 Gold (DE3) strain (Stratagene) in 1-liter flasks at 37°C with 500 ml of minimal M9 batch medium.

    Article Title: The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range
    Article Snippet: .. Protein expression and purification The proteins were expressed in the E. coli strain BL21 (DE3) RIL (Stratagene, La Jolla, USA) and purified slightly modified to remove the precursor tag according to standard protocols and stored in 40 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2 (standard buffer) at −80 °C until usage. .. For the NMR samples, minimal media containing 95% D2 O and supplemented with U-13 C glucose (Cambridge Isotope Laboratories, Tewksbury, USA) and 15 NH4 Cl (Cortecnet, Paris, France) were used for growing the cells and expressing the protein.

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: .. For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7. .. Expression was induced using 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) and cells were then grown overnight at 12o C. Cells were harvested and lysed with BugBuster® (EMDBiosciences) with protease inhibitors added [1 × complete EDTA-free protease inhibitor tablet (Roche)] before clearing by centrifugation (16,000 × g, 20 minutes, 4°C).

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: .. Preparation of Aβ42 for chemical kinetics experiments The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Article Title: New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis
    Article Snippet: GST, GST‐MoPdeH, GST‐MoPdeHEAL(AAA) , GST‐MoPdeHΔ HD , GST‐MoPdeHΔ HD+L1L2 , GST‐MoPdeHΔ HD+L1 , GST‐MoPdeHΔ HD+L2 , GST‐MoPdeHHD , GST‐MoPdeLL1L2 , GST‐MoPdeLL1 , GST‐MoPdeLL2 and GST‐MoPdeL were expressed in Escherichia coli BL21‐CodonPlus (DE3) cells (Stratagene, Cedar Creek, TX, USA). .. The target proteins were collected by sonication and further purified with glutathione sepharose beads incubated at 4 °C for 3 h. After centrifugation at 500 g for 5 min, glutathione sepharose beads were washed with washing buffer [20 m m Tris (pH 7.5), 0.25 m m NaCl, 2 m m EDTA, 2 m m ethyleneglycol‐bis(β‐aminoethylether)‐ N , N '‐tetraacetic acid (EGTA)], eluted with washing buffer containing reduced glutathione (pH 8.0) (the elution contained 5% glycerol), concentrated at 4 °C and stored at −70 °C.

    Dot Blot:

    Article Title: The funnel approach to the pre-crystallization production of membrane proteins
    Article Snippet: The initial expression studies for all clones utilized E. coli strain BL21 (DE3) GOLD™ (Stratagene). .. The His-tagged protein content of each culture well was analyzed using dot-blot techniques ( ).

    IA:

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: .. The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: .. Preparation of Aβ peptides The recombinant Aβ (M1-42) peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA), here called Aβ42, was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene) and purified as described previously with slight modifications ( ). .. Briefly, the purification procedure involved sonication of E. coli cells, dissolution of inclusion bodies in 8 M urea, ion exchange in batch mode on diethylaminoethyl cellulose resin, and lyophilization.

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: Preparation of Aβ peptides The recombinant Aβ (M1-42) peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA), here called Aβ42, was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene) and purified as described previously with slight modifications ( ). .. Isotopically labeled 15 N-Aβ42 was prepared by growing transformed E. coli BL21 Gold (DE3) strain (Stratagene) in 1-liter flasks at 37°C with 500 ml of minimal M9 batch medium.

    Article Title: Trodusquemine enhances Aβ42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes
    Article Snippet: .. Preparation of Aβ42 for chemical kinetics experiments The recombinant Aβ42 peptide (MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA) was expressed in the Escherichia coli BL21 Gold (DE3) strain (Stratagene, CA, USA) and purified as described previously . .. The purification procedure involved sonication of the E. coli cells, and a subsequent dissolution of the inclusion bodies in 8 M urea.

    Plasmid Preparation:

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA
    Article Snippet: .. The protein was expressed in E. coli BL21 (DE3)-Gold cells with the RIL-Codon plus plasmid (Stratagene). .. Cultures were grown at 37°C until the OD600 reached 0.5; the temperature was then shifted to 16°C, and isopropyl β-d -1-thiogalactopyranoside (IPTG) was added to 0.4 mM to induce expression.

    Article Title: Polymerase-IV occupancy at RNA-directed DNA methylation sites requires SHH1
    Article Snippet: .. The plasmid was transformed into the E. coli strain BL21 (DE3) RIL (Stratagene). .. The cells were cultured at 37 °C until the OD600 reached 0.8 and then the media was cooled to 20 °C and 0.2 mM IPTG was added to induce protein expression overnight.

    Article Title: The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix
    Article Snippet: .. Protein was expressed in Escherichia coli BL21(DE3)-Gold cells (Stratagene) with the RIL-Codon plus plasmid. ..

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: .. Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. After incubation with 0.1 mM IPTG at 15°C for 12 h, MUTYH-His6 protein was purified with TALON metal affinity resins (Clontech, Palo Alto, CA) and a TALON 2-ml disposable gravity column (Clontech).

    Article Title: Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance
    Article Snippet: .. The pET28a–lysdh, pet28a–aasadh and pET28a–lysdh–aasadh constructs and the empty vector were transformed into the BL21 DE3 pRIL E. coli strain (Stratagene, San Diego, CA, USA). ..

    Software:

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. The quality and concentration of MUTYH proteins were determined by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and Image J software (National Institutes of Health, Bethesda, MD).

    Protein Purification:

    Article Title: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation
    Article Snippet: Paragraph title: Protein purification ... For purification of GST-tagged eIF5 wt and mutant constructs, ArcticExpress™ BL21 DE3 RIL E. coli cells (Stratagene) were transformed with recombinant pGEX-4T-1 plasmids and then grown in LB medium to an A 600 of 0.7.

    Positron Emission Tomography:

    Article Title: The funnel approach to the pre-crystallization production of membrane proteins
    Article Snippet: Post induction, the pBAD constructs generally continued to grow normally, while the growth of many of the pET constructs stopped, with some cultures lysing. .. The initial expression studies for all clones utilized E. coli strain BL21 (DE3) GOLD™ (Stratagene).

    In Vitro:

    Article Title: New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis
    Article Snippet: Paragraph title: Detection of protein PDEase activity in vitro ... GST, GST‐MoPdeH, GST‐MoPdeHEAL(AAA) , GST‐MoPdeHΔ HD , GST‐MoPdeHΔ HD+L1L2 , GST‐MoPdeHΔ HD+L1 , GST‐MoPdeHΔ HD+L2 , GST‐MoPdeHHD , GST‐MoPdeLL1L2 , GST‐MoPdeLL1 , GST‐MoPdeLL2 and GST‐MoPdeL were expressed in Escherichia coli BL21‐CodonPlus (DE3) cells (Stratagene, Cedar Creek, TX, USA).

    Nuclear Magnetic Resonance:

    Article Title: The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range
    Article Snippet: Protein expression and purification The proteins were expressed in the E. coli strain BL21 (DE3) RIL (Stratagene, La Jolla, USA) and purified slightly modified to remove the precursor tag according to standard protocols and stored in 40 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2 (standard buffer) at −80 °C until usage. .. For the NMR samples, minimal media containing 95% D2 O and supplemented with U-13 C glucose (Cambridge Isotope Laboratories, Tewksbury, USA) and 15 NH4 Cl (Cortecnet, Paris, France) were used for growing the cells and expressing the protein.

    Concentration Assay:

    Article Title: Adenine DNA Glycosylase Activity of 14 Human MutY Homolog (MUTYH) Variant Proteins Found in Patients with Colorectal Polyposis and Cancer
    Article Snippet: Preparation of the recombinant MUTYH proteins E. coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) were transformed with the MUTYH-pET25b vector and cultured at 37°C until an A600 of 0.6. .. The quality and concentration of MUTYH proteins were determined by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and Image J software (National Institutes of Health, Bethesda, MD).

    Article Title: An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease
    Article Snippet: Isotopically labeled 15 N-Aβ42 was prepared by growing transformed E. coli BL21 Gold (DE3) strain (Stratagene) in 1-liter flasks at 37°C with 500 ml of minimal M9 batch medium. .. Isopropyl-β-d -thiogalactopyranoside was then added to a final concentration of 1 mM, and cells were grown at 37°C overnight.

    Article Title: The Alternative Sigma Factor ?B of Bacillus cereus: Response to Stress and Role in Heat Adaptation
    Article Snippet: E. coli BL21-Codonplus-(DE3)-RIL (Stratagene, La Jolla, Calif.) was used as the host for SigB overproduction. .. The antibiotics used were ampicillin at a concentration of 50 μg/ml, kanamycin at a concentration of 70 μg/ml, erythromycin at a concentration of 150 μg/ml (for E. coli ) or 5 μg/ml (for B. cereus ), spectinomycin at a concentration of 100 μg/ml, and polymyxin B at a concentration of 50 μg/ml for counterselection against E. coli upon conjugation.

    Article Title: Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals 1
    Article Snippet: Recombinant E. coli BL21 (DE3) Gold (Stratagene, La Jolla, CA) bacteria were plated in Luria-Bertani broth (LB)-agar containing 50 μg mL−1 ampicillin and 34 μg mL−1 chroramphenicol. .. Isopropylthio-β-galactoside was then added to a final concentration of 0.3 m m , and the cultures were grown for another 4 to 5 h at room temperature and aliquoted in 2-mL polypropylene tubes to 1.5-mL aliquots.

    Lysis:

    Article Title: New findings on phosphodiesterases, MoPdeH and MoPdeL, in Magnaporthe oryzae revealed by structural analysis
    Article Snippet: GST, GST‐MoPdeH, GST‐MoPdeHEAL(AAA) , GST‐MoPdeHΔ HD , GST‐MoPdeHΔ HD+L1L2 , GST‐MoPdeHΔ HD+L1 , GST‐MoPdeHΔ HD+L2 , GST‐MoPdeHHD , GST‐MoPdeLL1L2 , GST‐MoPdeLL1 , GST‐MoPdeLL2 and GST‐MoPdeL were expressed in Escherichia coli BL21‐CodonPlus (DE3) cells (Stratagene, Cedar Creek, TX, USA). .. Samples were induced with 0.1 m m IPTG (isopropyl‐β‐ d ‐1‐thiogalactopyranoside) at 18 °C for 4 h. After centrifugation at 2500 g for 10 min, the precipitated cells were resuspended in lysis buffer [1 × phosphate‐buffered saline (PBS), 10% glycerol].

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    Stratagene e coli protease deficient expression strain bl21
    Competitive binding of cannabinoid ligands on <t>BL21-107</t> membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.
    E Coli Protease Deficient Expression Strain Bl21, supplied by Stratagene, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli protease deficient expression strain bl21/product/Stratagene
    Average 70 stars, based on 1 article reviews
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    e coli protease deficient expression strain bl21 - by Bioz Stars, 2020-01
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    96
    Stratagene e coli bl21
    SDS-PAGE analysis of purified protein recombinant SllB in E. coli <t>BL21</t> cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.
    E Coli Bl21, supplied by Stratagene, used in various techniques. Bioz Stars score: 96/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21/product/Stratagene
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    91
    Stratagene e coli bl21 gold
    Interactions of MBP-UreD with other urease components in vivo. E. coli <t>BL21-Gold(DE3)</t> cells were cotransformed with either pEC002 (encoding MBP-UreD) or pMal-c2x (encoding MBP-LacZα) along with pEC004, pEC005, pEC006, pEC007, pEC008, or pEC009 (encoding UreABC, UreFG, UreABCEFG, UreE, UreF, or UreG, respectively). Soluble cell extracts were analyzed directly by SDS-PAGE (odd-numbered lanes) or subjected to amylose resin chromatography with the proteins eluted by maltose addition and analyzed by SDS-PAGE (even-numbered lanes). (A) MBP-UreD interactions with multiple urease components. (B) MBP-UreD interactions with single urease components. M, molecular mass markers.
    E Coli Bl21 Gold, supplied by Stratagene, used in various techniques. Bioz Stars score: 91/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Competitive binding of cannabinoid ligands on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Competitive binding of cannabinoid ligands on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Binding Assay, Expressing

    Ligand-binding assay on E. coli BL21-107 membranes

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Ligand-binding assay on E. coli BL21-107 membranes

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Ligand Binding Assay

    Time course of CB2-107 expression in E. coli . ( A ) An overnight culture of BL21-107 cells was used to inoculate several incubation flasks containing equal volumes of media. Induction was started when cell density reached OD 600 = 0.4. Cell samples were

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Time course of CB2-107 expression in E. coli . ( A ) An overnight culture of BL21-107 cells was used to inoculate several incubation flasks containing equal volumes of media. Induction was started when cell density reached OD 600 = 0.4. Cell samples were

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Expressing, Incubation

    Saturation binding of [ 3 H] CP-55,940 on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Saturation binding of [ 3 H] CP-55,940 on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Binding Assay, Expressing

    SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: SDS Page, Purification, Recombinant, Marker

    A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: Transmission Assay, Recombinant

    Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: Expressing, SDS Page, Marker, Western Blot, Recombinant

    Interactions of MBP-UreD with other urease components in vivo. E. coli BL21-Gold(DE3) cells were cotransformed with either pEC002 (encoding MBP-UreD) or pMal-c2x (encoding MBP-LacZα) along with pEC004, pEC005, pEC006, pEC007, pEC008, or pEC009 (encoding UreABC, UreFG, UreABCEFG, UreE, UreF, or UreG, respectively). Soluble cell extracts were analyzed directly by SDS-PAGE (odd-numbered lanes) or subjected to amylose resin chromatography with the proteins eluted by maltose addition and analyzed by SDS-PAGE (even-numbered lanes). (A) MBP-UreD interactions with multiple urease components. (B) MBP-UreD interactions with single urease components. M, molecular mass markers.

    Journal: Journal of Bacteriology

    Article Title: Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein ▿

    doi: 10.1128/JB.01426-09

    Figure Lengend Snippet: Interactions of MBP-UreD with other urease components in vivo. E. coli BL21-Gold(DE3) cells were cotransformed with either pEC002 (encoding MBP-UreD) or pMal-c2x (encoding MBP-LacZα) along with pEC004, pEC005, pEC006, pEC007, pEC008, or pEC009 (encoding UreABC, UreFG, UreABCEFG, UreE, UreF, or UreG, respectively). Soluble cell extracts were analyzed directly by SDS-PAGE (odd-numbered lanes) or subjected to amylose resin chromatography with the proteins eluted by maltose addition and analyzed by SDS-PAGE (even-numbered lanes). (A) MBP-UreD interactions with multiple urease components. (B) MBP-UreD interactions with single urease components. M, molecular mass markers.

    Article Snippet: In order to further investigate the interaction between UreD and urease apoprotein, soluble cell extracts of E. coli BL21-Gold(DE3)/pEC002 (containing MBP-UreD) and E. coli BL21-Gold(DE3)/pEC004 (containing UreABC) were mixed, incubated at room temperature for 1 h, and subjected to amylose resin pulldown analysis.

    Techniques: In Vivo, SDS Page, Chromatography

    Interactions of MBP-UreD with urease apoprotein. (A) In vivo complex formation. Soluble cell extracts of IPTG-induced E. coli BL21-Gold(DE3)/pEC002 (encoding MBP-UreD) and E. coli BL21-Gold(DE3)/pEC004 (encoding UreABC) were mixed, incubated at room temperature for 1 h, and chromatographed on amylose resin. Bound proteins were eluted in buffer containing 10 mM maltose and visualized by SDS-PAGE. Lanes: M, molecular mass marker; 1, pEC002 cell extracts; 2, pEC004 cell extracts; 3, coelution of UreABC with MBP-UreD from the amylose resin. (B) In vitro interactions. Purified MBP-UreD (2 μM) was mixed with isolated urease apoprotein (10 μM) in TEB buffer with 25 mM NaCl and, where indicated, cell extracts of E. coli MG1655. Reaction mixtures were incubated at room temperature for 1 h before the mix was subjected to amylose resin chromatography. Bound proteins were eluted with buffer containing 10 mM maltose. Lanes: M, molecular mass marker; 1, purified urease apoprotein; 2, purified MBP-UreD; 3, MBP-UreD plus urease apoprotein; 4, MBP-UreD plus urease apoprotein along with E. coli MG1655 cell extracts; 5, eluted fraction from lane 3; 6, eluted fraction from lane 4.

    Journal: Journal of Bacteriology

    Article Title: Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein ▿

    doi: 10.1128/JB.01426-09

    Figure Lengend Snippet: Interactions of MBP-UreD with urease apoprotein. (A) In vivo complex formation. Soluble cell extracts of IPTG-induced E. coli BL21-Gold(DE3)/pEC002 (encoding MBP-UreD) and E. coli BL21-Gold(DE3)/pEC004 (encoding UreABC) were mixed, incubated at room temperature for 1 h, and chromatographed on amylose resin. Bound proteins were eluted in buffer containing 10 mM maltose and visualized by SDS-PAGE. Lanes: M, molecular mass marker; 1, pEC002 cell extracts; 2, pEC004 cell extracts; 3, coelution of UreABC with MBP-UreD from the amylose resin. (B) In vitro interactions. Purified MBP-UreD (2 μM) was mixed with isolated urease apoprotein (10 μM) in TEB buffer with 25 mM NaCl and, where indicated, cell extracts of E. coli MG1655. Reaction mixtures were incubated at room temperature for 1 h before the mix was subjected to amylose resin chromatography. Bound proteins were eluted with buffer containing 10 mM maltose. Lanes: M, molecular mass marker; 1, purified urease apoprotein; 2, purified MBP-UreD; 3, MBP-UreD plus urease apoprotein; 4, MBP-UreD plus urease apoprotein along with E. coli MG1655 cell extracts; 5, eluted fraction from lane 3; 6, eluted fraction from lane 4.

    Article Snippet: In order to further investigate the interaction between UreD and urease apoprotein, soluble cell extracts of E. coli BL21-Gold(DE3)/pEC002 (containing MBP-UreD) and E. coli BL21-Gold(DE3)/pEC004 (containing UreABC) were mixed, incubated at room temperature for 1 h, and subjected to amylose resin pulldown analysis.

    Techniques: In Vivo, Incubation, SDS Page, Marker, In Vitro, Purification, Isolation, Chromatography