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Millipore protease inhibitor mixture
Protease Inhibitor Mixture, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/protease inhibitor mixture/product/Millipore
Average 99 stars, based on 36 article reviews
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protease inhibitor mixture - by Bioz Stars, 2022-09
99/100 stars

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  • 99
    Millipore co immunoprecipitation binding buffer
    EHD3 associates with Ca V 3.1 and Ca V 3.2. EHD3 Ig immobilized Protein A-conjugated agarose beads immunoprecipitates Ca V 3.1 ( A ) and Ca V 3.2 ( B ). C and D, EHD3 was included as a positive control. E , Na V 1.5 does not associate with EHD3 in <t>co-immunoprecipitation</t>
    Co Immunoprecipitation Binding Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/co immunoprecipitation binding buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    co immunoprecipitation binding buffer - by Bioz Stars, 2022-09
    99/100 stars
      Buy from Supplier

    98
    Millipore u87 mg glioma cells
    The RB1 gene and RB1 protein are expressed in RB116 cells. A : Multiplex ligation-dependent probe amplification RB1 (MLPA) is shown for RB116 cells. Gene dosage analysis shows a normal copy number (2) corresponding to all probes, with no duplications or deletions. Probes targeted 23 of RB1’s 27 exons and three nearby genes ( DLEU1, CHC1L, ITM2B ). Y79 cells, with a known multiexon deletion, served as a positive control. Normal human peripheral blood served as a negative control. B : Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis shows RB1 mRNA expression in RB116 cells. RB116 cells were evaluated alongside RB1-negative RB143 cells and RB1-positive MDA-MB231 breast cancer cells. RB116 cell expression was set at 1.0. The experiment was repeated three times and the error bars show standard deviation. No p values were calculated, as this was an experiment to determine presence or absence of RB1 and not meant to be comparative between cell lines. RB1 mRNA (mRNA) was detected in both RB116 and MDA-MB231 cells. C : western blot analysis detects RB1 protein in RB116 cells. Western immunoblotting was performed on RB116 cells and detected the expected p110 RB1 band that was identical to the positive controls (purified RB1 protein, MOLT4 human leukemia cells, and <t>U87</t> human glioma cells). No band was seen in the lane containing lysate from the negative control (RB1 negative RB143 cells). The blot was reprobed with alpha-tubulin to ensure equal protein loading of the cell lysates. D : Immunocytochemistry demonstrates perinuclear localization of RB1 protein in RB116 cells. RB116 cells were compared with the negative control (Y79 cells) and the positive control (MDA-MB231 cells). The experiment was repeated three times. Note the perinuclear localization of RB1 in RB116 cells. A control slide of RB116 cells received an isotype control antibody instead of the anti-RB1 antibody.
    U87 Mg Glioma Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/u87 mg glioma cells/product/Millipore
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    u87 mg glioma cells - by Bioz Stars, 2022-09
    98/100 stars
      Buy from Supplier

    93
    Millipore bugbuster master mix
    Solubility of the N-terminal RyR2 fragments. (A) and (B) RyR2 1–247 ·His 6 , RyR2 391–606 ·His 6 , RyR2 409–606 ·His 6 and RyR2 1–606 ·His 6 solubilized with <t>BugBuster</t> and Triton X-100, respectively. (C) Nus·RyR2 1–606 and Nus·RyR2 230–606 solubilized with Triton X-100 and (D) Trx·RyR2 384–606 ·His 6 , Trx·RyR2 391–606 ·His 6 and Trx·RyR2 409–606 ·His 6 solubilized with Triton X-100. SF and CD represent eluates from the Ni-NTA resin and the insoluble cell debris, respectively. Arrowheads indicate the positions of the respective protein bands.
    Bugbuster Master Mix, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bugbuster master mix/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bugbuster master mix - by Bioz Stars, 2022-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    EHD3 associates with Ca V 3.1 and Ca V 3.2. EHD3 Ig immobilized Protein A-conjugated agarose beads immunoprecipitates Ca V 3.1 ( A ) and Ca V 3.2 ( B ). C and D, EHD3 was included as a positive control. E , Na V 1.5 does not associate with EHD3 in co-immunoprecipitation

    Journal: The Journal of Biological Chemistry

    Article Title: Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria *

    doi: 10.1074/jbc.M115.646893

    Figure Lengend Snippet: EHD3 associates with Ca V 3.1 and Ca V 3.2. EHD3 Ig immobilized Protein A-conjugated agarose beads immunoprecipitates Ca V 3.1 ( A ) and Ca V 3.2 ( B ). C and D, EHD3 was included as a positive control. E , Na V 1.5 does not associate with EHD3 in co-immunoprecipitation

    Article Snippet: Protein A-conjugated agarose beads (AffiGel; Bio-Rad) were incubated with either control mouse Ig or anti-EHD3 Ig in co-immunoprecipitation binding buffer (PBS with 0.1% Triton X-100 and protease inhibitor mixture (Sigma)) for 12 h at 4 °C.

    Techniques: Positive Control, Immunoprecipitation

    DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in complete medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: DAG treatment causes S/G 2 phase cell cycle arrest and induces replication-dependent DNA damage. a , b Serum-starved (ST for 24 h) M059K and PC-3 cells were treated with or without 5 μM DAG in complete medium for the indicated periods of time. Then the cells were collected for cell cycle analysis using PI staining as described in “Materials and Methods”. The representative flow cytometric histograms from two individual experiments are shown with the corresponding percentages of cell populations at G 0 /G 1 , S, and G 2 /M phases. c PC-3 cells were synchronized at G 0 /G 1 phase by 24 h serum starvation before treatment with 50 μM DAG for 1 h in complete medium. After the treatment, DAG was removed and the cells were continued to incubate in complete medium for another 24 h (DAG 1 h + WO 24 h). WO stands for washout. After that, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 (green) and anti-ɣH2AX (red) antibodies. Representative IF images are shown from two independent experiments. The scale bar stands for 10 μm. d From each experimental condition in c , we examined 100–120 cells and calculated the percentages of cyclin A2-/ɣH2AX- (double negative) and cyclin A2 + /ɣH2AX + (double positive) populations of cells. The corresponding statistical analysis shows significance (** p ≤ 0.01; *** p ≤ 0.001). e PC-3 and M059K cells were treated with 20 μM DAG for 48 h or 50 μM DAG for 1 h (in quiescent cells by 24 h serum starvation) followed by 24 h incubation without the drug in complete medium (DAG 1 h + WO 24 h). DSBs were analyzed by neutral comet assay and representative images are shown. The scale bar stands for 50 μm. f Tail moment from 50–100 cells at each experimental condition in e was analyzed by ImageJ (OpenComet) software and shows significance compare to untreated cells (** p ≤ 0.01; **** p ≤ 0.0001). g , h PC-3 or M059K cells were seeded in 96-well plates and cultured for 24 h in either serum-deprived medium or complete medium. Then the cells were treated with different concentrations of DAG for 72 h followed by crystal violet assay. Cell survival rates compared to untreated condition were shown as mean ± standard deviation.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Cell Cycle Assay, Staining, Incubation, Neutral Comet Assay, Software, Cell Culture, Crystal Violet Assay, Standard Deviation

    DAG-induced DNA damage activates HR pathway. a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: DAG-induced DNA damage activates HR pathway. a PC-3 and M059K cells were synchronized at G 0 /G 1 phase by serum starvation for 24 h. The quiescent cells were treated with 50 μM DAG for 1 h followed by washout and additional incubation of 0, 20, 24, or 48 h in complete medium. Cell lysates were then analyzed for HR mediators by western blot from three independent experiments. b Quiescent PC-3 cells were treated with 50 μM DAG for 1 h followed by incubation without the drug for 24 h. Cells were pre-extracted by cytoskeletal buffer, fixed, permeabilized, and probed with corresponding antibodies for indicated proteins. Representative confocal images are shown from three independent experiments. The scale bar represents 5 μm. c PC-3 (80–150 cells) from each condition in b were quantified for foci-positive cells with statistical analysis (** p ≤ 0.01; *** p ≤ 0.001). d , e PC-3 or M059K cells were knocked down of BRCA1 by transient transfection using non-overlapping BRCA1-targeting siRNAs (B1, siBRCA1–2; B2, siBRCA1–14; B3, siBRCA1–25; B4, siBRCA1–15; or B5, siBRCA1–17). The negative control siRNA (C) was also included. After 24 h transfection, the cells were treated with different concentrations of DAG (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days followed by crystal violet assay. The IC 50 values of DAG in control or BRCA1 knockdown cells were calculated using GraphPad Prism 6. The data are presented as mean ± standard deviation from three independent experiments with statistical significance (* p ≤ 0.05; ** p ≤ 0.01). In parallel western blot analysis confirmed BRCA1 knockdown. f Cell lysates were extracted from two pairs of isogenic colorectal cancer cell lines. Western blot analysis showed the expression of MLH1 and MSH2 in HCT116-PS-MLH1 and LoVo-PS-MSH2 cells, respectively, from three independent experiments. g The two pairs of isogenic colorectal cancer cells were treated with different concentrations of DAG for 3 days followed by crystal violet assay. The data in the curve are presented as mean ± standard deviation from three independent experiments.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Incubation, Western Blot, Transfection, Negative Control, Crystal Violet Assay, Standard Deviation, Expressing

    Cytotoxic effect of DAG in GBM and PCa cell lines. a M059K and PC-3 cells were treated with or without 10 μM DAG in complete medium for 72 h. The representative bright-field images were shown and the scale bar represents 100 μm. b , c Two GBM (M059K and M059J) and four PCa (PC-3, LNCaP, DU-145, and PC-3-DR) cell lines were treated with different concentrations of DAG (0, 50 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 72 h in 96-well culture plates. Following the treatment, crystal violet assay was performed and the IC 50 values of DAG for each cell line were calculated by fitting a sigmoidal dose-response curve to the data using GraphPad Prism 6. The data on the curve are presented as mean ± standard deviation. Three individual experiments were performed for each cell line.

    Journal: Cell Death & Disease

    Article Title: Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

    doi: 10.1038/s41419-020-02780-8

    Figure Lengend Snippet: Cytotoxic effect of DAG in GBM and PCa cell lines. a M059K and PC-3 cells were treated with or without 10 μM DAG in complete medium for 72 h. The representative bright-field images were shown and the scale bar represents 100 μm. b , c Two GBM (M059K and M059J) and four PCa (PC-3, LNCaP, DU-145, and PC-3-DR) cell lines were treated with different concentrations of DAG (0, 50 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 72 h in 96-well culture plates. Following the treatment, crystal violet assay was performed and the IC 50 values of DAG for each cell line were calculated by fitting a sigmoidal dose-response curve to the data using GraphPad Prism 6. The data on the curve are presented as mean ± standard deviation. Three individual experiments were performed for each cell line.

    Article Snippet: Western blot analysisCells were lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid; EDTA) supplemented with PhosSTOP phosphatase inhibitor and cOmplete protease inhibitor (MilliporeSigma, Oakville, Canada).

    Techniques: Crystal Violet Assay, Standard Deviation

    The RB1 gene and RB1 protein are expressed in RB116 cells. A : Multiplex ligation-dependent probe amplification RB1 (MLPA) is shown for RB116 cells. Gene dosage analysis shows a normal copy number (2) corresponding to all probes, with no duplications or deletions. Probes targeted 23 of RB1’s 27 exons and three nearby genes ( DLEU1, CHC1L, ITM2B ). Y79 cells, with a known multiexon deletion, served as a positive control. Normal human peripheral blood served as a negative control. B : Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis shows RB1 mRNA expression in RB116 cells. RB116 cells were evaluated alongside RB1-negative RB143 cells and RB1-positive MDA-MB231 breast cancer cells. RB116 cell expression was set at 1.0. The experiment was repeated three times and the error bars show standard deviation. No p values were calculated, as this was an experiment to determine presence or absence of RB1 and not meant to be comparative between cell lines. RB1 mRNA (mRNA) was detected in both RB116 and MDA-MB231 cells. C : western blot analysis detects RB1 protein in RB116 cells. Western immunoblotting was performed on RB116 cells and detected the expected p110 RB1 band that was identical to the positive controls (purified RB1 protein, MOLT4 human leukemia cells, and U87 human glioma cells). No band was seen in the lane containing lysate from the negative control (RB1 negative RB143 cells). The blot was reprobed with alpha-tubulin to ensure equal protein loading of the cell lysates. D : Immunocytochemistry demonstrates perinuclear localization of RB1 protein in RB116 cells. RB116 cells were compared with the negative control (Y79 cells) and the positive control (MDA-MB231 cells). The experiment was repeated three times. Note the perinuclear localization of RB1 in RB116 cells. A control slide of RB116 cells received an isotype control antibody instead of the anti-RB1 antibody.

    Journal: Molecular Vision

    Article Title: RB116: An RB1+ retinoblastoma cell line expressing primitive markers

    doi:

    Figure Lengend Snippet: The RB1 gene and RB1 protein are expressed in RB116 cells. A : Multiplex ligation-dependent probe amplification RB1 (MLPA) is shown for RB116 cells. Gene dosage analysis shows a normal copy number (2) corresponding to all probes, with no duplications or deletions. Probes targeted 23 of RB1’s 27 exons and three nearby genes ( DLEU1, CHC1L, ITM2B ). Y79 cells, with a known multiexon deletion, served as a positive control. Normal human peripheral blood served as a negative control. B : Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis shows RB1 mRNA expression in RB116 cells. RB116 cells were evaluated alongside RB1-negative RB143 cells and RB1-positive MDA-MB231 breast cancer cells. RB116 cell expression was set at 1.0. The experiment was repeated three times and the error bars show standard deviation. No p values were calculated, as this was an experiment to determine presence or absence of RB1 and not meant to be comparative between cell lines. RB1 mRNA (mRNA) was detected in both RB116 and MDA-MB231 cells. C : western blot analysis detects RB1 protein in RB116 cells. Western immunoblotting was performed on RB116 cells and detected the expected p110 RB1 band that was identical to the positive controls (purified RB1 protein, MOLT4 human leukemia cells, and U87 human glioma cells). No band was seen in the lane containing lysate from the negative control (RB1 negative RB143 cells). The blot was reprobed with alpha-tubulin to ensure equal protein loading of the cell lysates. D : Immunocytochemistry demonstrates perinuclear localization of RB1 protein in RB116 cells. RB116 cells were compared with the negative control (Y79 cells) and the positive control (MDA-MB231 cells). The experiment was repeated three times. Note the perinuclear localization of RB1 in RB116 cells. A control slide of RB116 cells received an isotype control antibody instead of the anti-RB1 antibody.

    Article Snippet: Sodium dodecyl sulfate PAGE and western blotting RB116, Y79, and U87-MG glioma cells were pelleted and lysed in 2x Laemmli buffer (125 mM Tris-Hcl), 10% glycerol, 10% sodium dodecyl sulfate) with 1% Sigma protease inhibitor cocktail (#P8340; Sigma-Aldrich, St. Louis, MO), boiled for 10 min, centrifuged 10 min, at 14,000 g. Protein concentrations were determined with the Bio-Rad (Hercules, CA) DC assay (#500–001) and proteins separated by sodium dodecyl sulfate PAGE using a 4%–12% Bis-Tris Novex NuPage gel (Invitrogen # NP0322BOX; Life Technologies) run for 90 min at 200 V, with 20 μg total protein loaded per lane, versus MW size standard (Amersham #RPN-800; GE Healthcare Lifesciences, Pittsburgh, PA).

    Techniques: Multiplex Assay, Ligation, Amplification, Multiplex Ligation-dependent Probe Amplification, Positive Control, Negative Control, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Multiple Displacement Amplification, Standard Deviation, Western Blot, Purification, Immunocytochemistry

    Solubility of the N-terminal RyR2 fragments. (A) and (B) RyR2 1–247 ·His 6 , RyR2 391–606 ·His 6 , RyR2 409–606 ·His 6 and RyR2 1–606 ·His 6 solubilized with BugBuster and Triton X-100, respectively. (C) Nus·RyR2 1–606 and Nus·RyR2 230–606 solubilized with Triton X-100 and (D) Trx·RyR2 384–606 ·His 6 , Trx·RyR2 391–606 ·His 6 and Trx·RyR2 409–606 ·His 6 solubilized with Triton X-100. SF and CD represent eluates from the Ni-NTA resin and the insoluble cell debris, respectively. Arrowheads indicate the positions of the respective protein bands.

    Journal: Protein Expression and Purification

    Article Title: Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2

    doi: 10.1016/j.pep.2009.12.014

    Figure Lengend Snippet: Solubility of the N-terminal RyR2 fragments. (A) and (B) RyR2 1–247 ·His 6 , RyR2 391–606 ·His 6 , RyR2 409–606 ·His 6 and RyR2 1–606 ·His 6 solubilized with BugBuster and Triton X-100, respectively. (C) Nus·RyR2 1–606 and Nus·RyR2 230–606 solubilized with Triton X-100 and (D) Trx·RyR2 384–606 ·His 6 , Trx·RyR2 391–606 ·His 6 and Trx·RyR2 409–606 ·His 6 solubilized with Triton X-100. SF and CD represent eluates from the Ni-NTA resin and the insoluble cell debris, respectively. Arrowheads indicate the positions of the respective protein bands.

    Article Snippet: Cell lysis, protein extraction and purification For obtaining the soluble recombinant protein, the following protocols were used: (a) About 0.1 g of frozen cells (obtained from 20 ml of culture) was homogenized in 500 μl of BugBuster Master Mix (Novagen – Merck Biosciences, Darmstadt, Germany) Protease inhibitor Mix B (Serva, Nordmark, Germany) and incubated on ice for 30 min to obtain an optimal lysis of the cells. (b) About 0.1 g of frozen cells homogenized in 500 μl of 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 7 mM β-mercaptoethanol containing protease inhibitor Mix B was sonicated for 15 s, then 60 μl of 10% Triton X-100 was added and stirred for 1 h at 4 °C.

    Techniques: Solubility