e coli bl21  (Thermo Fisher)


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    Structured Review

    Thermo Fisher e coli bl21
    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli <t>BL21</t> (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    e coli bl21 - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis"

    Article Title: SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

    Journal: Molecular Cancer

    doi: 10.1186/s12943-017-0724-6

    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin
    Figure Legend Snippet: KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin

    Techniques Used: Modification, In Vitro, Transfection, Western Blot, Plasmid Preparation, Transformation Assay, Purification, Stripping Membranes, Mutagenesis, Construct

    2) Product Images from "Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection"

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    Journal: ACS Omega

    doi: 10.1021/acsomega.0c01181

    Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.
    Figure Legend Snippet: Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Techniques Used: DNA Extraction, Lysis, Electrophoresis, Polymerase Chain Reaction, Amplification, Incubation

    Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.
    Figure Legend Snippet: Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Techniques Used: Fluorescence, Microscopy, DNA Extraction, Polymerase Chain Reaction

    DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).
    Figure Legend Snippet: DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Techniques Used: DNA Extraction, Polymerase Chain Reaction, Amplification, DNA Binding Assay, Binding Assay, Purification, Genetically Modified, Transgenic Assay, Modification

    QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.
    Figure Legend Snippet: QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    3) Product Images from "Recombinant lipidated Zika virus envelope protein domain III elicits durable neutralizing antibody responses against Zika virus in mice"

    Article Title: Recombinant lipidated Zika virus envelope protein domain III elicits durable neutralizing antibody responses against Zika virus in mice

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-020-00646-x

    Production and purification of recombinant Zika virus envelope protein domain III (rZE3) and recombinant lipidated Zika virus envelope protein domain III (rLZE3). The plasmid maps of pZE3 ( a ) and pLZE3 ( d ) for the production of rZE3 and rLZE3, respectively. The purification of rZE3 ( b , c ) and rLZE3 ( e , f ) was monitored by 10% reducing Tricine-SDS-PAGE followed by Coomassie Blue staining and immunoblotting with anti-His-tag antibodies. rZE3 and rLZE3 were expressed in the E. coli strains BL21 (DE3) and C43 (DE3), respectively. Lanes 1, 5, 9, and 13: protein expression without IPTG induction; lanes 2, 6, 10, and 14: protein expression after IPTG induction; lanes 3 and 7: extraction of rZE3 from inclusion bodies; lanes 11 and 15: soluble fraction of rLZE3; and lanes 4, 8, 12, and 16: purified proteins. Lanes 5–8 and lanes 13–16 show the induction and purification processes for rZE3 and rLZE3, respectively, evaluated by immunoblotting. The arrows show the electrophoretic positions of rZE3 or rLZE3. g Mass spectrum analysis of rLZE3. The N-terminus of the rLZE3 fragments was obtained by trypsin digestion and further examined with a WatersR MALDI micro MX™ mass spectrometer. MALDI-TOF MS spectra revealed lipid peptide signals with three m/z value peaks of 1452.129, 1466.144, and 1480.160
    Figure Legend Snippet: Production and purification of recombinant Zika virus envelope protein domain III (rZE3) and recombinant lipidated Zika virus envelope protein domain III (rLZE3). The plasmid maps of pZE3 ( a ) and pLZE3 ( d ) for the production of rZE3 and rLZE3, respectively. The purification of rZE3 ( b , c ) and rLZE3 ( e , f ) was monitored by 10% reducing Tricine-SDS-PAGE followed by Coomassie Blue staining and immunoblotting with anti-His-tag antibodies. rZE3 and rLZE3 were expressed in the E. coli strains BL21 (DE3) and C43 (DE3), respectively. Lanes 1, 5, 9, and 13: protein expression without IPTG induction; lanes 2, 6, 10, and 14: protein expression after IPTG induction; lanes 3 and 7: extraction of rZE3 from inclusion bodies; lanes 11 and 15: soluble fraction of rLZE3; and lanes 4, 8, 12, and 16: purified proteins. Lanes 5–8 and lanes 13–16 show the induction and purification processes for rZE3 and rLZE3, respectively, evaluated by immunoblotting. The arrows show the electrophoretic positions of rZE3 or rLZE3. g Mass spectrum analysis of rLZE3. The N-terminus of the rLZE3 fragments was obtained by trypsin digestion and further examined with a WatersR MALDI micro MX™ mass spectrometer. MALDI-TOF MS spectra revealed lipid peptide signals with three m/z value peaks of 1452.129, 1466.144, and 1480.160

    Techniques Used: Purification, Recombinant, Plasmid Preparation, SDS Page, Staining, Expressing, Mass Spectrometry

    4) Product Images from "The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection"

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.810440

    Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p
    Figure Legend Snippet: Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p

    Techniques Used: Recombinant, Activity Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Purification, SDS Page, Molecular Weight, Negative Control, Western Blot

    5) Product Images from "Effects of the cyclophilin-type peptidylprolyl cis-trans isomerase from Pyropia yezoensis against hydrogen peroxide-induced oxidative stress in HepG2 cells"

    Article Title: Effects of the cyclophilin-type peptidylprolyl cis-trans isomerase from Pyropia yezoensis against hydrogen peroxide-induced oxidative stress in HepG2 cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.6517

    Purification and identification of recombinant PPI protein. (A) SDS-PAGE analysis of PETppi transformed E.coli BL21 (DE3) and purified recombinant PPI. Lane 1, protein marker; lane 2, crude extract of pETppi transformed E. coli BL21 (DE3) prior to induction; lane 3, crude extract of pETppi transformed E. coli BL21 (DE3) after induction with 1 mM IPTG for 4 h; lane 4, purified recombinant PPI protein. (B) ESI-Q-TOF MS of purified recombinant PPI protein. The partial sequences of 4 peaks from the recombinant PPI were identified by MS/MS (Arrow).
    Figure Legend Snippet: Purification and identification of recombinant PPI protein. (A) SDS-PAGE analysis of PETppi transformed E.coli BL21 (DE3) and purified recombinant PPI. Lane 1, protein marker; lane 2, crude extract of pETppi transformed E. coli BL21 (DE3) prior to induction; lane 3, crude extract of pETppi transformed E. coli BL21 (DE3) after induction with 1 mM IPTG for 4 h; lane 4, purified recombinant PPI protein. (B) ESI-Q-TOF MS of purified recombinant PPI protein. The partial sequences of 4 peaks from the recombinant PPI were identified by MS/MS (Arrow).

    Techniques Used: Purification, Recombinant, SDS Page, Transformation Assay, Marker, Mass Spectrometry

    6) Product Images from "The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection"

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.810440

    Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p
    Figure Legend Snippet: Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p

    Techniques Used: Recombinant, Activity Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Purification, SDS Page, Molecular Weight, Negative Control, Western Blot

    7) Product Images from "The Intracellular Citrus Huanglongbing Bacterium, 'Candidatus Liberibacter asiaticus' Encodes Two Novel Autotransporters"

    Article Title: The Intracellular Citrus Huanglongbing Bacterium, 'Candidatus Liberibacter asiaticus' Encodes Two Novel Autotransporters

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068921

    Expression and outer membrane localization of a novel autotransporter protein, LasA I , of ‘ Candidatus Liberibacter asiaticus’ (Las). SDS-PAGE ( A ) and Western blot ( B ) analysis of E. coli containing the pET102- lasA I construct. M: A molecular mass marker; lane 1: Whole-cell lysate from E. coli BL21 containing plasmid pET102 alone; lane 2: Whole-cell lysate from E. coli BL21 containing recombinant plasmid pET102- lasA I, lane 3: outer membrane protein, lane 4: purified LasA I from whole-cell pellet, lane 5: LasA I protein without proteinase K treatment, Lane 6: LasA I with proteinase K treatment.
    Figure Legend Snippet: Expression and outer membrane localization of a novel autotransporter protein, LasA I , of ‘ Candidatus Liberibacter asiaticus’ (Las). SDS-PAGE ( A ) and Western blot ( B ) analysis of E. coli containing the pET102- lasA I construct. M: A molecular mass marker; lane 1: Whole-cell lysate from E. coli BL21 containing plasmid pET102 alone; lane 2: Whole-cell lysate from E. coli BL21 containing recombinant plasmid pET102- lasA I, lane 3: outer membrane protein, lane 4: purified LasA I from whole-cell pellet, lane 5: LasA I protein without proteinase K treatment, Lane 6: LasA I with proteinase K treatment.

    Techniques Used: Expressing, SDS Page, Western Blot, Construct, Marker, Plasmid Preparation, Recombinant, Purification

    8) Product Images from "Chimeric epitope vaccine against Leptospira interrogans infection and induced specific immunity in guinea pigs"

    Article Title: Chimeric epitope vaccine against Leptospira interrogans infection and induced specific immunity in guinea pigs

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0852-y

    Characterization of the expressed and purified chimeric r4R protein. a SDS-PAGE analysis of the expression and purification steps of the r4R protein. Pellet (lane 1 and 3) and supernatant (lane 2 and 4) fractions of lysates from E. coli BL21 (DE3) cells containing the empty vector pET28a (lane 1 and 2) or pET28a-4R (lane 3 and 4) were electrophoresed in a 10 % SDS-PAGE gel. M indicates the protein ladder. Lane 5 contains the purified recombinant 4R protein. b Anti- Leptospira Western blot analysis. The purified chimeric r4R protein was run on a SDS-PAGE gel and transferred to a PVDF membrane. Serum from heat-killed L. interrogans strain Lai immunized rabbits was used as a primary antibody to detect r4R. Serum from PBS injected rabbits was used as control. c Anti-r4R Western analysis. The purified chimeric r4R protein was run on SDS-PAGE gel and transferred to a PVDF membrane. Serum from guinea pigsimmunized with PBS (negative control) or chimeric protein, were used as primary antibody
    Figure Legend Snippet: Characterization of the expressed and purified chimeric r4R protein. a SDS-PAGE analysis of the expression and purification steps of the r4R protein. Pellet (lane 1 and 3) and supernatant (lane 2 and 4) fractions of lysates from E. coli BL21 (DE3) cells containing the empty vector pET28a (lane 1 and 2) or pET28a-4R (lane 3 and 4) were electrophoresed in a 10 % SDS-PAGE gel. M indicates the protein ladder. Lane 5 contains the purified recombinant 4R protein. b Anti- Leptospira Western blot analysis. The purified chimeric r4R protein was run on a SDS-PAGE gel and transferred to a PVDF membrane. Serum from heat-killed L. interrogans strain Lai immunized rabbits was used as a primary antibody to detect r4R. Serum from PBS injected rabbits was used as control. c Anti-r4R Western analysis. The purified chimeric r4R protein was run on SDS-PAGE gel and transferred to a PVDF membrane. Serum from guinea pigsimmunized with PBS (negative control) or chimeric protein, were used as primary antibody

    Techniques Used: Purification, SDS Page, Expressing, Plasmid Preparation, Recombinant, Western Blot, Injection, Negative Control

    9) Product Images from "A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli"

    Article Title: A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-019-10145-1

    Controlled induction of sfGFP expression. a Fluorescence quantification of sfGFP production with increasing amounts of rhamnose in shaker flasks at different time points: pre-induction (white), 5 h (gray), or 7 h (black) after induction. The E. coli pSAR-2 strain without sfGFP insert is indicated as “Empty”. Error bars indicate standard deviations of two biological replicates. b Flow cytometry analysis (FACS) data of E. coli BL21 cells with pSAR-2 empty and pSAR-2::sfGFP vector expressing sfGFP, 4 h after induction with different amounts of rhamnose in shaker flasks. Left plots showing cell granularity by forward scatter (FSC-A, x -axis) and side scatter (SSC-A, y -axis) per cell. Right histograms showing the number of cells per mean GFP fluorescence intensity (au). c Scalability of sfGFP expression in different culturing systems induced at OD 600nm 0.6–0.8 with 10 mM rhamnose
    Figure Legend Snippet: Controlled induction of sfGFP expression. a Fluorescence quantification of sfGFP production with increasing amounts of rhamnose in shaker flasks at different time points: pre-induction (white), 5 h (gray), or 7 h (black) after induction. The E. coli pSAR-2 strain without sfGFP insert is indicated as “Empty”. Error bars indicate standard deviations of two biological replicates. b Flow cytometry analysis (FACS) data of E. coli BL21 cells with pSAR-2 empty and pSAR-2::sfGFP vector expressing sfGFP, 4 h after induction with different amounts of rhamnose in shaker flasks. Left plots showing cell granularity by forward scatter (FSC-A, x -axis) and side scatter (SSC-A, y -axis) per cell. Right histograms showing the number of cells per mean GFP fluorescence intensity (au). c Scalability of sfGFP expression in different culturing systems induced at OD 600nm 0.6–0.8 with 10 mM rhamnose

    Techniques Used: Expressing, Fluorescence, Flow Cytometry, Cytometry, FACS, Plasmid Preparation

    SDS-PAGE and Western blot analysis of antibody affinity chromatography purification of PGT135 scFv. Analysis of feed, flow-through (FT), wash, and elution fractions 8–11 (E8–E11) of an affinity chromatography run using Capto-L for purification of PGT135 scFv antibody fragment from the soluble protein fraction of E. coli BL21 cells expressing PGT135 scFv from pSAR-2 vector. a Representative SDS-PAGE gel showing total protein in different analyzed fractions and PGT135 scFv antibody with an expected band size of 29 kDa. Elution fractions show highly enriched PGT135 scFv after purification. b Representative western blot with HRP–Protein L binding to PGT135 scFv in different analyzed fractions. In the first lane, 2.8 μg of commercially available His-tagged PGT135 scFv (scFv-His 6 xHis) is included for semi-quantification. PGT135 scFv is clearly visible and semi quantified in feed and elution fractions
    Figure Legend Snippet: SDS-PAGE and Western blot analysis of antibody affinity chromatography purification of PGT135 scFv. Analysis of feed, flow-through (FT), wash, and elution fractions 8–11 (E8–E11) of an affinity chromatography run using Capto-L for purification of PGT135 scFv antibody fragment from the soluble protein fraction of E. coli BL21 cells expressing PGT135 scFv from pSAR-2 vector. a Representative SDS-PAGE gel showing total protein in different analyzed fractions and PGT135 scFv antibody with an expected band size of 29 kDa. Elution fractions show highly enriched PGT135 scFv after purification. b Representative western blot with HRP–Protein L binding to PGT135 scFv in different analyzed fractions. In the first lane, 2.8 μg of commercially available His-tagged PGT135 scFv (scFv-His 6 xHis) is included for semi-quantification. PGT135 scFv is clearly visible and semi quantified in feed and elution fractions

    Techniques Used: SDS Page, Western Blot, Affinity Chromatography, Purification, Flow Cytometry, Expressing, Plasmid Preparation, Binding Assay

    Expression of antibody fragment PGT135 scFv. PGT135 scFv is expressed by E. coli BL21 from the pSAR-2:scFv vector with different concentrations of rhamnose for induction, and at two different temperatures (30 °C and 25 °C) and harvest times (21 h and 48 h). a Representative western blot with Protein L–HRP conjugate binding to PGT135 scFv antibody fragments in the soluble protein fraction. Numbers 3, 10, and 15 indicate the amount of rhamnose added for induction in mM. E. coli BL21 pSAR-2 empty induced with 10 mM of rhamnose is included as a negative control. Two different known concentrations of the commercially available His 6 -tagged antibody PGT135 scFv-6xHis are included for quantification. b Graph showing semi-quantitative data for PGT135 scFv production in shaker flasks as determined by western blot based on two biological replicates. Black bars show PGT135 scFv in total cell fraction and gray bars in soluble protein fraction. Error bars indicate standard deviation of two biological duplicates
    Figure Legend Snippet: Expression of antibody fragment PGT135 scFv. PGT135 scFv is expressed by E. coli BL21 from the pSAR-2:scFv vector with different concentrations of rhamnose for induction, and at two different temperatures (30 °C and 25 °C) and harvest times (21 h and 48 h). a Representative western blot with Protein L–HRP conjugate binding to PGT135 scFv antibody fragments in the soluble protein fraction. Numbers 3, 10, and 15 indicate the amount of rhamnose added for induction in mM. E. coli BL21 pSAR-2 empty induced with 10 mM of rhamnose is included as a negative control. Two different known concentrations of the commercially available His 6 -tagged antibody PGT135 scFv-6xHis are included for quantification. b Graph showing semi-quantitative data for PGT135 scFv production in shaker flasks as determined by western blot based on two biological replicates. Black bars show PGT135 scFv in total cell fraction and gray bars in soluble protein fraction. Error bars indicate standard deviation of two biological duplicates

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Binding Assay, Negative Control, Standard Deviation

    10) Product Images from "Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs"

    Article Title: Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054938

    AP2µ Binds to Isolated CD79a but not CD79b. Panel A , Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. Panel B , AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. Panel C , Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. Panel D , The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in BL21 E. coli cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to in vitro translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL) 3 , and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL) 3 , fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL) 3 –AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.
    Figure Legend Snippet: AP2µ Binds to Isolated CD79a but not CD79b. Panel A , Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. Panel B , AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. Panel C , Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. Panel D , The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in BL21 E. coli cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to in vitro translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL) 3 , and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL) 3 , fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL) 3 –AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.

    Techniques Used: Isolation, Binding Assay, Activation Assay, Positive Control, Negative Control, In Vitro, Labeling

    11) Product Images from "Gut Microbiota Abrogates Anti-α-Gal IgA Response in Lungs and Protects against Experimental Aspergillus Infection in Poultry"

    Article Title: Gut Microbiota Abrogates Anti-α-Gal IgA Response in Lungs and Protects against Experimental Aspergillus Infection in Poultry

    Journal: Vaccines

    doi: 10.3390/vaccines8020285

    Oral administration of E. coli O86:B7 induces a significant decrease in the levels of anti-Galα1-3Galβ1-4GlcNAc IgY Abs in A. fumigatus -infected turkeys. The levels of circulating anti-α-Gal IgY Abs to Galα1-3Gal ( A ) and Galα1-3Galβ1-4GlcNAc ( B ) were measured by ELISA. Anti-Galα1-3Gal IgY Abs increased in the sera of turkeys treated with E. coli O86:B7 and E. coli BL21. Oral administration of E. coli O86:B7 produces a significant reduction in anti-Galα1-3Galβ1-4GlcNAc IgY Abs when compared with turkeys that were treated or not E. coli BL21. Results shown are means and standard deviation values. Results were compared by One-way ANOVA with Dunnett’s multiple comparison test applied for individual comparisons (* p
    Figure Legend Snippet: Oral administration of E. coli O86:B7 induces a significant decrease in the levels of anti-Galα1-3Galβ1-4GlcNAc IgY Abs in A. fumigatus -infected turkeys. The levels of circulating anti-α-Gal IgY Abs to Galα1-3Gal ( A ) and Galα1-3Galβ1-4GlcNAc ( B ) were measured by ELISA. Anti-Galα1-3Gal IgY Abs increased in the sera of turkeys treated with E. coli O86:B7 and E. coli BL21. Oral administration of E. coli O86:B7 produces a significant reduction in anti-Galα1-3Galβ1-4GlcNAc IgY Abs when compared with turkeys that were treated or not E. coli BL21. Results shown are means and standard deviation values. Results were compared by One-way ANOVA with Dunnett’s multiple comparison test applied for individual comparisons (* p

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Oral administration of E. coli O86:B7 protects turkeys against aspergillosis. Clinical examination revealed that A. fumigatus infection produces open-mouthed breathing (OMB) in turkeys treated with PBS or E. coli BL21. Turkeys treated with E. coli O86:B7 were protected from developing OMB ( A ). Pulmonary lesions (i.e., granulomas, delimited area and white arrow heads) were scored (see methods). Examples of lungs with scores 0 to 3 are shown ( B ). Granuloma score was lower in turkeys treated with E. coli O86:B7 ( C ). Lung samples were processed for histopathology and stained with hematoxylin-eosin-saffron (HES, D ) and periodic acid-schiff (PAS, E ). Histological lesions were scored (see methods). Examples of histopathology samples with scores 0 to 3 are shown. Visible peribronchial regions (asterisk) and granulomas (delimited area and black arrow heads) are shown (HES score, D ). The presence of fungal germ-tube/hyphae (black arrows) and mycelium (white arrows) was scored (see methods). Granulomas associated with fungal hyphae (delimited area and black arrow heads) are shown (PAS score, E ). HES and PAS scores were lower in turkeys treated with E. coli O86:B7 ( F , G ). The presence of viable Aspergillus in lungs was quantified by colony-forming unit (CFU) counting assay ( H ). Fungal DNA levels were measured by A. fumigatus -specific 28S qPCR normalizing against turkey actb and gapdh as host genes using the 2 −ΔΔ C t ratio method. Results are relative to 28S levels in the control group (i.e., PBS) ( I ). No significant change was observed in the amount of CFU and 28S fold change ( H , I ). Size of bars is indicated. 100X magnification. Results shown are means and standard deviation values. Results were compared by One-way ANOVA with Dunnett’s multiple comparison test applied for individual comparisons (*** p
    Figure Legend Snippet: Oral administration of E. coli O86:B7 protects turkeys against aspergillosis. Clinical examination revealed that A. fumigatus infection produces open-mouthed breathing (OMB) in turkeys treated with PBS or E. coli BL21. Turkeys treated with E. coli O86:B7 were protected from developing OMB ( A ). Pulmonary lesions (i.e., granulomas, delimited area and white arrow heads) were scored (see methods). Examples of lungs with scores 0 to 3 are shown ( B ). Granuloma score was lower in turkeys treated with E. coli O86:B7 ( C ). Lung samples were processed for histopathology and stained with hematoxylin-eosin-saffron (HES, D ) and periodic acid-schiff (PAS, E ). Histological lesions were scored (see methods). Examples of histopathology samples with scores 0 to 3 are shown. Visible peribronchial regions (asterisk) and granulomas (delimited area and black arrow heads) are shown (HES score, D ). The presence of fungal germ-tube/hyphae (black arrows) and mycelium (white arrows) was scored (see methods). Granulomas associated with fungal hyphae (delimited area and black arrow heads) are shown (PAS score, E ). HES and PAS scores were lower in turkeys treated with E. coli O86:B7 ( F , G ). The presence of viable Aspergillus in lungs was quantified by colony-forming unit (CFU) counting assay ( H ). Fungal DNA levels were measured by A. fumigatus -specific 28S qPCR normalizing against turkey actb and gapdh as host genes using the 2 −ΔΔ C t ratio method. Results are relative to 28S levels in the control group (i.e., PBS) ( I ). No significant change was observed in the amount of CFU and 28S fold change ( H , I ). Size of bars is indicated. 100X magnification. Results shown are means and standard deviation values. Results were compared by One-way ANOVA with Dunnett’s multiple comparison test applied for individual comparisons (*** p

    Techniques Used: Infection, Histopathology, Staining, Real-time Polymerase Chain Reaction, Standard Deviation

    Expression of turkey and chicken cytokine genes in response to oral administration of E. coli O86:B7 and E. coli BL21 and α-Gal-BSA immunization. The figure displays the mRNA expression levels of INFγ , IL6 , IL2 , IL10 and MyD88 in ceca ( A ) and lungs ( B ) of turkeys and IL6 and IL2 in lungs of chicken ( C ). Total RNA was extracted and gene expression levels were measured by qPCR normalizing against turkey actb and gapdh as housekeeping genes, using the using the 2 −ΔΔ C t ratio method. Expression levels are relative to the control group (i.e., PBS). Results shown are means and standard deviation values. Results were compared by One-way ANOVA with Dunnett’s multiple comparison test applied for individual comparisons (* p
    Figure Legend Snippet: Expression of turkey and chicken cytokine genes in response to oral administration of E. coli O86:B7 and E. coli BL21 and α-Gal-BSA immunization. The figure displays the mRNA expression levels of INFγ , IL6 , IL2 , IL10 and MyD88 in ceca ( A ) and lungs ( B ) of turkeys and IL6 and IL2 in lungs of chicken ( C ). Total RNA was extracted and gene expression levels were measured by qPCR normalizing against turkey actb and gapdh as housekeeping genes, using the using the 2 −ΔΔ C t ratio method. Expression levels are relative to the control group (i.e., PBS). Results shown are means and standard deviation values. Results were compared by One-way ANOVA with Dunnett’s multiple comparison test applied for individual comparisons (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    12) Product Images from "Engineered fungal polyketide biosynthesis in Pichia pastoris: a potential excellent host for polyketide production"

    Article Title: Engineered fungal polyketide biosynthesis in Pichia pastoris: a potential excellent host for polyketide production

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-12-77

    Nucleotide and amino acid sequences of ACP amd SDS-PAGE of PPTase and ACP and ACPm. (A) nucleotide and amino acid sequences ACP domain of citrinin polyketide synthase. The conserved serine at site 56 which marked by square frame was mutated by overlap PCR using primers mutant5/mutant3 to generate ACPm. (B) SDS-PAGE of PPTase expressed by GS115-NpgA-HIS 6 ; (C) SDS-PAGE of ACP and ACPm expressed by E. coli BL21. Lane 1: Lysate supernatant of GS115-NpgA-HIS 6 ; Lane 2: Flow-through fraction of GS115-NpgA-HIS 6 ; Lane 3: Eluted protein of GS115-NpgA-HIS 6 ; Lane 4: Lysate supernatant of wild type E. coli BL21 strain as negative control; Lane 5: Lysate supernatant of BL21-ACP; Lane 6: Lysate supernatant of BL21-ACPm. M: Protein marker. Protein purification were described in the Section Protein expression and purification in Methods. The arrows indicates the objective proteins.
    Figure Legend Snippet: Nucleotide and amino acid sequences of ACP amd SDS-PAGE of PPTase and ACP and ACPm. (A) nucleotide and amino acid sequences ACP domain of citrinin polyketide synthase. The conserved serine at site 56 which marked by square frame was mutated by overlap PCR using primers mutant5/mutant3 to generate ACPm. (B) SDS-PAGE of PPTase expressed by GS115-NpgA-HIS 6 ; (C) SDS-PAGE of ACP and ACPm expressed by E. coli BL21. Lane 1: Lysate supernatant of GS115-NpgA-HIS 6 ; Lane 2: Flow-through fraction of GS115-NpgA-HIS 6 ; Lane 3: Eluted protein of GS115-NpgA-HIS 6 ; Lane 4: Lysate supernatant of wild type E. coli BL21 strain as negative control; Lane 5: Lysate supernatant of BL21-ACP; Lane 6: Lysate supernatant of BL21-ACPm. M: Protein marker. Protein purification were described in the Section Protein expression and purification in Methods. The arrows indicates the objective proteins.

    Techniques Used: SDS Page, Polymerase Chain Reaction, Flow Cytometry, Negative Control, Marker, Protein Purification, Expressing, Purification

    13) Product Images from "The Serine Protease Autotransporters TagB, TagC, and Sha from Extraintestinal Pathogenic Escherichia coli Are Internalized by Human Bladder Epithelial Cells and Cause Actin Cytoskeletal Disruption"

    Article Title: The Serine Protease Autotransporters TagB, TagC, and Sha from Extraintestinal Pathogenic Escherichia coli Are Internalized by Human Bladder Epithelial Cells and Cause Actin Cytoskeletal Disruption

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21093047

    The serine catalytic site is necessary for the cytopathic effect of TagB and TagC. ( A ) Concentrated supernatants containing 30 μg of protein per well derived from E. coli BL21 clones expressing TagB, TagC, Sha, or their respective serine mutant variant proteins were incubated with monolayers of the 5637 human bladder epithelial cell line for 5 h at 37 °C. Cytopathic effects (white triangle) were absent in cells treated with the serine catalytic site mutant variants of TagB or TagC. The empty vector (pBCsk+) without insert was used as a negative control. The scale bar represents 20 µm. ( B ) Cytotoxicity measured by LDH release from 5637 human bladder cells after incubation with supernatant filtrates of different clones (30 μg of protein per well) at 37 °C for 5 h. Empty vector (pBCsk+) was used as a negative control and maximum LDH release (positive control) was determined by treatment with lysis solution. Data are the means of three independent experiments, and error bars represent the standard errors of the means. Significant differences between lysis caused by native and mutant SPATEs were determined using Student’s t -test with *** p
    Figure Legend Snippet: The serine catalytic site is necessary for the cytopathic effect of TagB and TagC. ( A ) Concentrated supernatants containing 30 μg of protein per well derived from E. coli BL21 clones expressing TagB, TagC, Sha, or their respective serine mutant variant proteins were incubated with monolayers of the 5637 human bladder epithelial cell line for 5 h at 37 °C. Cytopathic effects (white triangle) were absent in cells treated with the serine catalytic site mutant variants of TagB or TagC. The empty vector (pBCsk+) without insert was used as a negative control. The scale bar represents 20 µm. ( B ) Cytotoxicity measured by LDH release from 5637 human bladder cells after incubation with supernatant filtrates of different clones (30 μg of protein per well) at 37 °C for 5 h. Empty vector (pBCsk+) was used as a negative control and maximum LDH release (positive control) was determined by treatment with lysis solution. Data are the means of three independent experiments, and error bars represent the standard errors of the means. Significant differences between lysis caused by native and mutant SPATEs were determined using Student’s t -test with *** p

    Techniques Used: Derivative Assay, Clone Assay, Expressing, Mutagenesis, Variant Assay, Incubation, Plasmid Preparation, Negative Control, Positive Control, Lysis

    The autoaggregation phenotype is independent of the serine protease motif. Clones of E. coli BL21 expressing TagB, TagC, Sha, or their respective serine-site mutants were grown 18 h and adjusted to an OD 600 of 1.5 and left to rest at 4 °C. Samples were taken at 1 cm from the top surface of the cultures after 3 h to determine the change in OD 600 . Assays were performed in triplicate, and the rate of autoaggregation was determined by the mean decrease in OD 600nm after 3 h. E. coli BL21 pBCsk+ vector without insert (empty vector) was used as a negative control and the AIDA-1 autotransporter was the positive control for autoaggregation. Error bars represent standard errors of the means (*** p
    Figure Legend Snippet: The autoaggregation phenotype is independent of the serine protease motif. Clones of E. coli BL21 expressing TagB, TagC, Sha, or their respective serine-site mutants were grown 18 h and adjusted to an OD 600 of 1.5 and left to rest at 4 °C. Samples were taken at 1 cm from the top surface of the cultures after 3 h to determine the change in OD 600 . Assays were performed in triplicate, and the rate of autoaggregation was determined by the mean decrease in OD 600nm after 3 h. E. coli BL21 pBCsk+ vector without insert (empty vector) was used as a negative control and the AIDA-1 autotransporter was the positive control for autoaggregation. Error bars represent standard errors of the means (*** p

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Negative Control, Positive Control

    ( A ) Silver stained SDS-PAGE analysis of concentrated supernatants of E. coli BL21 expressing SPATE proteins. Filtered supernatants from clones expressing TagB, TagC, and Sha or the variant TagB S255A, TagC S252A, and Sha S258A proteins were concentrated through Amicon filters with a 50 kDa cutoff. Samples containing 5 µg of protein were migrated and stained with silver stain. ( B – E ) Immunogold Electron Microscopy (EM) of SPATEs (Serine protease autotransporters of Enterobacteriaceae ) localized to the outer membrane and extracellular medium. Immunogold-TEM micrographs of SPATEs using SPATE-specific antiserum. Bacteria were cultured to 0.6 OD 600nm in Luria-Bertani medium. E. coli BL21 pBCsk+ expressing TagB ( B ), TagC ( C ), and Sha ( D ) labelled with immunogold particles. ( E ) E. coli BL21 pBcsk+ (vector only control) shows no immunogold staining. Insets represents boxed areas of higher magnification showing clustering of SPATE proteins. All images were acquired at ×17,000 magnification; scale bars represent 1 µm, and 0.5 µm (Insets).
    Figure Legend Snippet: ( A ) Silver stained SDS-PAGE analysis of concentrated supernatants of E. coli BL21 expressing SPATE proteins. Filtered supernatants from clones expressing TagB, TagC, and Sha or the variant TagB S255A, TagC S252A, and Sha S258A proteins were concentrated through Amicon filters with a 50 kDa cutoff. Samples containing 5 µg of protein were migrated and stained with silver stain. ( B – E ) Immunogold Electron Microscopy (EM) of SPATEs (Serine protease autotransporters of Enterobacteriaceae ) localized to the outer membrane and extracellular medium. Immunogold-TEM micrographs of SPATEs using SPATE-specific antiserum. Bacteria were cultured to 0.6 OD 600nm in Luria-Bertani medium. E. coli BL21 pBCsk+ expressing TagB ( B ), TagC ( C ), and Sha ( D ) labelled with immunogold particles. ( E ) E. coli BL21 pBcsk+ (vector only control) shows no immunogold staining. Insets represents boxed areas of higher magnification showing clustering of SPATE proteins. All images were acquired at ×17,000 magnification; scale bars represent 1 µm, and 0.5 µm (Insets).

    Techniques Used: Staining, SDS Page, Expressing, Clone Assay, Variant Assay, Silver Staining, Electron Microscopy, Transmission Electron Microscopy, Cell Culture, Plasmid Preparation

    Effects of TagB, TagC, and Sha on the actin cytoskeleton of bladder epithelial cells is serine-protease-motif dependent. ( A ) Concentrated supernatant extracts (30 μg of protein per well) from E. coli BL21 clones expressing TagB, TagC, or Sha and their respective serine catalytic site mutants were incubated with monolayers of human bladder (5637) epithelial cells for 5 h at 37 °C. After incubation, cells were fixed and permeabilized. Actin was stained with fluorescently labeled phalloidin (green) and the nucleus was stained by DAPI (blue). Cells treated with the filtered supernatant of E. coli BL21 pBCsk+ without insert (empty vector) were used as a negative control. Slides were observed by confocal microscopy. Inset images from the left panels are magnified in the panels to the right. Bars represent 10 µm. ( B ) Quantitative analysis of fluorescence intensity of F-actin. Analysis of fluorescent intensity was done at the original magnification by measuring the mean gray value with ImageJ software [ 22 ] with an n value of at least 10 cells. Data values represent the mean ± SEM of at least three independent experiments. (* p
    Figure Legend Snippet: Effects of TagB, TagC, and Sha on the actin cytoskeleton of bladder epithelial cells is serine-protease-motif dependent. ( A ) Concentrated supernatant extracts (30 μg of protein per well) from E. coli BL21 clones expressing TagB, TagC, or Sha and their respective serine catalytic site mutants were incubated with monolayers of human bladder (5637) epithelial cells for 5 h at 37 °C. After incubation, cells were fixed and permeabilized. Actin was stained with fluorescently labeled phalloidin (green) and the nucleus was stained by DAPI (blue). Cells treated with the filtered supernatant of E. coli BL21 pBCsk+ without insert (empty vector) were used as a negative control. Slides were observed by confocal microscopy. Inset images from the left panels are magnified in the panels to the right. Bars represent 10 µm. ( B ) Quantitative analysis of fluorescence intensity of F-actin. Analysis of fluorescent intensity was done at the original magnification by measuring the mean gray value with ImageJ software [ 22 ] with an n value of at least 10 cells. Data values represent the mean ± SEM of at least three independent experiments. (* p

    Techniques Used: Clone Assay, Expressing, Incubation, Staining, Labeling, Plasmid Preparation, Negative Control, Confocal Microscopy, Fluorescence, Software

    14) Product Images from "Expression of Melittin in Fusion with GST in Escherichia coli and Its Purification as a Pure Peptide with Good Bacteriostatic Efficacy"

    Article Title: Expression of Melittin in Fusion with GST in Escherichia coli and Its Purification as a Pure Peptide with Good Bacteriostatic Efficacy

    Journal: ACS Omega

    doi: 10.1021/acsomega.0c00085

    (A) Tris-SDS-PAGE analysis of purification of GST-MET and GST with glutathione-affinity chromatography; M, Protein MW Marker (Low); Lanes 1 and 2, soluble proteins from E. coli BL21/pGEX-6p-1 and E. coli BL21/pGEX-MET; Lanes 3 and 4, the elution of GST and GST-MET by 10 mM reduced glutathione elutes GST; (B) The digestion of GST-MET with different concentrations of PPase. Lane 1, GST-MET before the digestion; Lanes 2–5, the digestion of GST-MET under the treatment of different concentration ratios of GST-MET and PPase: 10:1, 5:1, 2.5:1, and 1:1. (C) Tricine-SDS-PAGE analysis of MET purification with glutathione sepharose high-performance medium. M1, protein molecular weight marker (Low); M2, ultralow molecular weight protein marker; Lane 1, before digestion; Lane 2, the purified MET.
    Figure Legend Snippet: (A) Tris-SDS-PAGE analysis of purification of GST-MET and GST with glutathione-affinity chromatography; M, Protein MW Marker (Low); Lanes 1 and 2, soluble proteins from E. coli BL21/pGEX-6p-1 and E. coli BL21/pGEX-MET; Lanes 3 and 4, the elution of GST and GST-MET by 10 mM reduced glutathione elutes GST; (B) The digestion of GST-MET with different concentrations of PPase. Lane 1, GST-MET before the digestion; Lanes 2–5, the digestion of GST-MET under the treatment of different concentration ratios of GST-MET and PPase: 10:1, 5:1, 2.5:1, and 1:1. (C) Tricine-SDS-PAGE analysis of MET purification with glutathione sepharose high-performance medium. M1, protein molecular weight marker (Low); M2, ultralow molecular weight protein marker; Lane 1, before digestion; Lane 2, the purified MET.

    Techniques Used: SDS Page, Purification, Affinity Chromatography, Marker, Concentration Assay, Molecular Weight

    (A) Expression of proMET in E. coli BL21/pGEX-proMET. M, Protein Molecular Weight Marker (Low); Lane 1, total proteins of E. coli BL21/pGEX-6p-1 before induction; Lanes 2 and 3, soluble and insoluble proteins of E. coli BL21/pGEX-6p-1 after induction by 0.1 mM IPTG; Lanes 4–6, total, soluble, and insoluble proteins from E. coli BL21/pGEX-proMET after induction by 0.1 mM IPTG. (B) Expression of MET in E. coli BL21/pGEX-MET. M, Protein Molecular Weight Marker (Low); Lane 1, total protein from E. coli BL21/pGEX-MET before induction; Lanes 2–4: total, soluble, and precipitated proteins of E. coli BL21/pGEX-MET after induction by 0.1 mM IPTG.
    Figure Legend Snippet: (A) Expression of proMET in E. coli BL21/pGEX-proMET. M, Protein Molecular Weight Marker (Low); Lane 1, total proteins of E. coli BL21/pGEX-6p-1 before induction; Lanes 2 and 3, soluble and insoluble proteins of E. coli BL21/pGEX-6p-1 after induction by 0.1 mM IPTG; Lanes 4–6, total, soluble, and insoluble proteins from E. coli BL21/pGEX-proMET after induction by 0.1 mM IPTG. (B) Expression of MET in E. coli BL21/pGEX-MET. M, Protein Molecular Weight Marker (Low); Lane 1, total protein from E. coli BL21/pGEX-MET before induction; Lanes 2–4: total, soluble, and precipitated proteins of E. coli BL21/pGEX-MET after induction by 0.1 mM IPTG.

    Techniques Used: Expressing, Molecular Weight, Marker

    15) Product Images from "Characterization and Functional Analysis of Four Glutathione S-Transferases from the Migratory Locust, Locusta migratoria"

    Article Title: Characterization and Functional Analysis of Four Glutathione S-Transferases from the Migratory Locust, Locusta migratoria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058410

    Analysis of the heterologously expressed and purified recombinant L. migratoria GST proteins by SDS-PAGE. (A) LmGSTd1, (B) LmGSTs5, (C) LmGSTt1, (D) LmGSTu1. The gel (12%) was stained with Coomassie Blue G-250. Lane M, protein molecular size marker. Lane 1, extract of BL21/JM109 carrying the expression vector for GSTs without IPTG. Lane 2, extract of BL21/JM109 carrying the expression vector for GSTs with IPTG induction. Lane 3, purified locust GSTs.
    Figure Legend Snippet: Analysis of the heterologously expressed and purified recombinant L. migratoria GST proteins by SDS-PAGE. (A) LmGSTd1, (B) LmGSTs5, (C) LmGSTt1, (D) LmGSTu1. The gel (12%) was stained with Coomassie Blue G-250. Lane M, protein molecular size marker. Lane 1, extract of BL21/JM109 carrying the expression vector for GSTs without IPTG. Lane 2, extract of BL21/JM109 carrying the expression vector for GSTs with IPTG induction. Lane 3, purified locust GSTs.

    Techniques Used: Purification, Recombinant, SDS Page, Staining, Marker, Expressing, Plasmid Preparation

    16) Product Images from "Functional characterization and expression analysis of rice δ1-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism"

    Article Title: Functional characterization and expression analysis of rice δ1-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00591

    Expression of rice P5C dehydrogenase in E. coli and affinity purification . A truncated version of rice P5C dehydrogenase lacking the predicted mitochondrial transit peptide (Supplementary Figure 1 ) was expressed in E. coli , strain BL21 DE3 pLysS. Cells were analyzed by SDS-PAGE before and after the induction with 1 mM IPTG (A) . Six hours after IPTG addition, cells were harvested and extracted, and the plant protein (⇨) was purified by affinity chromatography on a His-Select™ Nickel Gel column (B) . The molecular mass of protein standards is indicated.
    Figure Legend Snippet: Expression of rice P5C dehydrogenase in E. coli and affinity purification . A truncated version of rice P5C dehydrogenase lacking the predicted mitochondrial transit peptide (Supplementary Figure 1 ) was expressed in E. coli , strain BL21 DE3 pLysS. Cells were analyzed by SDS-PAGE before and after the induction with 1 mM IPTG (A) . Six hours after IPTG addition, cells were harvested and extracted, and the plant protein (⇨) was purified by affinity chromatography on a His-Select™ Nickel Gel column (B) . The molecular mass of protein standards is indicated.

    Techniques Used: Expressing, Affinity Purification, SDS Page, Purification, Affinity Chromatography

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    Amplification:

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    Construct:

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    Positron Emission Tomography:

    Article Title: Molecular Characterization of Fluoroquinolone Resistance in Mycobacterium tuberculosis: Functional Analysis of gyrA Mutation at Position 74 ▿ Mutation at Position 74 ▿ †
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    Expressing:

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  • 92
    Thermo Fisher e coli bl21 cells
    SDS-PAGE analysis and activity of nitrite reductase expressed by E. coli <t>BL21.</t> (a) M: protein marker; Lane 1: protein product. (b) Nitrite reductase activity of E. coli BL21 transformed via OMVs under different temperatures.
    E Coli Bl21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21 cells/product/Thermo Fisher
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    94
    Thermo Fisher e coli bl21
    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli <t>BL21</t> (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher plasmid interference assay bl21 ai
    Transcription-dependent loss of the target plasmid. <t>BL21-AI</t> strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.
    Plasmid Interference Assay Bl21 Ai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SDS-PAGE analysis and activity of nitrite reductase expressed by E. coli BL21. (a) M: protein marker; Lane 1: protein product. (b) Nitrite reductase activity of E. coli BL21 transformed via OMVs under different temperatures.

    Journal: bioRxiv

    Article Title: Outer Membrane Vesicles Mediated Horizontal Transfer of an Aerobic Denitrification Gene between Escherichia coli

    doi: 10.1101/835694

    Figure Lengend Snippet: SDS-PAGE analysis and activity of nitrite reductase expressed by E. coli BL21. (a) M: protein marker; Lane 1: protein product. (b) Nitrite reductase activity of E. coli BL21 transformed via OMVs under different temperatures.

    Article Snippet: E. coli BL21 cells were stained with VybrantTM DiO live cell tracer (ThermoFisher).

    Techniques: SDS Page, Activity Assay, Marker, Transformation Assay

    The LCSM images of E. coli BL21 induced by IPTG. PH: phase contrast image; Green fluorescence: green fluorescence image; Overlay: image of phase contrast image and green fluorescence image superimposed.

    Journal: bioRxiv

    Article Title: Outer Membrane Vesicles Mediated Horizontal Transfer of an Aerobic Denitrification Gene between Escherichia coli

    doi: 10.1101/835694

    Figure Lengend Snippet: The LCSM images of E. coli BL21 induced by IPTG. PH: phase contrast image; Green fluorescence: green fluorescence image; Overlay: image of phase contrast image and green fluorescence image superimposed.

    Article Snippet: E. coli BL21 cells were stained with VybrantTM DiO live cell tracer (ThermoFisher).

    Techniques: Confocal Laser Scanning Microscopy, Fluorescence

    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin

    Journal: Molecular Cancer

    Article Title: SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

    doi: 10.1186/s12943-017-0724-6

    Figure Lengend Snippet: KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin

    Article Snippet: Briefly, pGEX-4T-1-KHSRP-WT was co-expressed with or without pE1E2SUMO1 plasmid in E.coli BL21 (DE3) respectively, and then lysed by using B-PER Protein Extraction Reagent (#78248, Thermo Fisher, USA) and incubated wi th Glutathione sepharose 4B (GE healthcare) at 4 °C overnight.

    Techniques: Modification, In Vitro, Transfection, Western Blot, Plasmid Preparation, Transformation Assay, Purification, Stripping Membranes, Mutagenesis, Construct

    Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: Optimization of DNA extraction with fPAMMPs. (A) DNA extraction by incubating fPAMMPs with E. coli BL21 lysis for various times. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs only, (2–6) fPAMMPs incubated with E. coli BL21 lysis for (2) 1 min, (3) 2 min, (4) 5 min, (5) 10 min, and (6) 20 min. (B) DNA extraction by just incubating fPAMMPs with E. coli BL21 lysis (a, b) for 30 sand (c, d) for 15 s. (a, c) Electrophoresis of fPAMMPs. (b, d) PCR amplification of the T7 RNA polymerase gene. (1) fPAMMPs and (2) fPAMMP@BL21 DNA. (C) DNA extraction from different amounts of cells and PCR amplification. OD 600 of bacterial culture was measured, and 2 × 10 9 , 1 × 10 9 , 5 × 10 8 , 2.5 × 10 8 , and 1.25 × 10 8 cfu of cells (from right to left) were used for DNA extraction. (a) Electrophoresis of fPAMMPs. (b) PCR amplification of E. coli 16S rDNA with fPAMMP@DNA. (c) PCR amplification of the E. coli T7 RNA polymerase gene with fPAMMP@DNA. (D) PCR amplification of target genes, 16S rDNA and T7 RNA polymerase gene, from fPAMMP@DNA that were kept at different conditions (−80, −20, and −4 °C) for various times.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: DNA Extraction, Lysis, Electrophoresis, Polymerase Chain Reaction, Amplification, Incubation

    Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: Preparation of fluorescence-free fPAMMPs (called as PAMMPs). (A) Microscopy images of fPAMMPs before and after NaBH 4 reduction. From right to left, light field, green VF, red VF, and blue VF. Scale bars are 200 μm. (B) Images of fPAMMPs and PAMMPs with SEM. The scale bars from left to right are 100, 20, 20, and 10 μm. (C) NIRF image of fPAMMPs before and after NaBH 4 reduction. (D) DNA extraction and PCR detection with PAMMP. (a) DNA extraction with PAMMP. (1) PAMMPs and (2–4) PAMMP@BL21 DNA. In DNA extraction, (2) 1.25 × 10 8 , (3) 2.5 × 10 8 , and (4) 5 × 10 8 cfu of cells were used. (b) PCR detection with PAMMP@DNA. (1) PAMMPs, (2) PAMMP@DH5α DNA extracted with 5 × 10 8 cfu of cells, and (3–5) PAMMP@BL21 DNA extracted with (3) 1.25 × 10 8 , (4) 2.5 × 10 8 , and (5) 5 × 10 8 cfu of cells.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: Fluorescence, Microscopy, DNA Extraction, Polymerase Chain Reaction

    DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: DNA extraction and direct PCR amplification using fPAMMPs. (A) DNA binding assay. (a) fPAMMPs binding with the purified free DNA. (1) fPAMMP@SiHa gDNA, (2) fPAMMPs, and (3) free SiHa gDNA. fPAMMP@SiHa gDNA, 2 μg SiHa gDNA was mixed with 80 μL of fPAMMP. The fPAMMP@SiHa gDNA was washed three times with water and resuspended in 50 μL of water wherein 20 μL of which was then loaded in the gel. Free SiHa gDNA, 200 ng loading. (b) Extraction of gDNA from E. coli BL21 and DH5α with fPAMMPs. (c) Subsequent PCR amplification of a 165 bp fragment of the T7 RNA polymerase gene that is contained by BL21 but not by DH5α. The various fPAMMPs in panel b were used as the PCR amplification template. (1) fPAMMPs, (2) fPAMMP@DH5α DNA, and (3) fPAMMP@BL21 DNA. (B) Extraction of gDNA from more various samples with fPAMMPs and detected fPAMMP@DNA with PCR. (a) Mouse liver tissue from which fragments of RELA and GAPDH genes were amplified. (1) NTC (for GAPDH), (2) NTC (for RELA), (3) GAPDH, and (4) RELA. (b) Human cell (left), solid tissue (middle), and blood plasma (right) from which five STR and GAPDH genes were amplified. (1) NTC, (2) GAPDH, (3) GATA193H05, (4) D11S4951, (5) D2S2951, (6) D6S2421, and (7) D11S4957. (c) Human plasma from which a fragment of the TERT promoter was amplified. (1) NTC and (2) TERT. (d) Plant leaf tissue from which NOS and zSSllb genes were amplified. The NOS gene is contained by GMP but not contained by NGMP, and the zSSllb gene is the plant house-keeping gene. (1) zSSllb in NGMP, (2) zSSllb in GMP, (3) NOS in NGMP, and (4) NOS in GMP. NTC, no template control (fPAMMPs only); GMP, genetically modified plant (i.e., transgenic plant); and NGMP, nongenetically modified plant (i.e., nontransgenic plant).

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: DNA Extraction, Polymerase Chain Reaction, Amplification, DNA Binding Assay, Binding Assay, Purification, Genetically Modified, Transgenic Assay, Modification

    QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Journal: ACS Omega

    Article Title: Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection

    doi: 10.1021/acsomega.0c01181

    Figure Lengend Snippet: QPCR detection of the T7 RNA polymerase gene with PAMMP@DNA and fPAMMP@DNA. (A, B) QPCR detection with (A) PAMMP@DNA and (B) fPAMMP@DNA. The amplification plots and melt curves of standards and samples were provided. The copy numbers of different samples were calculated with the standard curve and provided as numbers on the standard curve. (1) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of BL21 culture (start culture), (2) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 10 time-diluted start culture, (3) PAMMP/fPAMMP@BL21 DNA extracted with 50 μL of 100 time-diluted start culture, (4) PAMMP/fPAMMP@DH5α DNA extracted with 50 μL of DH5α culture, and (5) PAMMP/fPAMMP.

    Article Snippet: Bacteria The T7 RNA polymerase gene and 16S rDNA were detected with fPAMMP@DNA of E. coli BL21 and DH5α.

    Techniques: Real-time Polymerase Chain Reaction, Amplification

    Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Article Snippet: Plasmid interference assay BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis

    Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Article Snippet: Plasmid interference assay BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Transformation Assay