mouse monoclonal anti gp64  (Millipore)


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    Structured Review

    Millipore mouse monoclonal anti gp64
    Ac109 localization in virions and in Sf-9 cells. (A) Western blot analysis of purified virions. Budded viruses (BVs), obtained from supernatants of Sf-9 cells infected with wild type (wt) Ac MNPV at a multiplicity of infection (MOI) of 0.1, were purified through a 25% w/v sucrose followed by a 25–65% sucrose gradient. Purified BVs were resuspended in PBS, and nucleocapsids and envelopes were separated using 1% IGEPAL and ultracentrifugation through a glycerol cushion. Occlusion derived viruses (ODVs) were obtained after alkaline treatment of polyhedra recovered from the infected Sf-9 cells. ODVs were purified through a discontinuous 30–65% sucrose gradient. Ac109, <t>GP64</t> and VP39 proteins were detected from purified ODVs, BVs, BVs envelopes (Env) and nucleocapsid (Nc) fractions with a polyclonal serum anti Ac109-GST fusion protein, an anti-GP64 monoclonal antibody or an anti-VP39 monoclonal antibody. Sf-9 cells infected with wt Ac MNPV (IC) were used as a control of Ac109 detection. (B) Subcellular localization of Ac109 in living cells. Ac109 fused to YFP protein at either C or N-terminus (Ac109:YFP and YFP:Ac109) and transcribed from pOpIE2 promoter was detected in Sf-9.RNuc cells by confocal microscopy at 48 h post-transfection (hpt). (C) Kinetics of subcellular localization of Ac109 during baculovirus infection. Confocal microscopy of Sf-9.RNuc cells infected with Acppolp109-109Y virus at a MOI of 5 is shown at indicated h post-infection (hpi). Ac109 was fused to YFP at the C-terminal end (Ac109:YFP) and transcribed from the ac109 promoter. (D) Living insect cells infected with Acppolp109-109Y virus showing a heterogeneous Ac109:YFP accumulation around forming polyhedra in the nucleus. (E) Ac109:YFP fluorescence concentrated in the already formed polyhedra. (F) Cytoplasmic accumulations of Ac109:YFP in Sf-9.RNuc cells at different stages of viral infection. Bars represent 10 µm.
    Mouse Monoclonal Anti Gp64, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1868 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny"

    Article Title: AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046146

    Ac109 localization in virions and in Sf-9 cells. (A) Western blot analysis of purified virions. Budded viruses (BVs), obtained from supernatants of Sf-9 cells infected with wild type (wt) Ac MNPV at a multiplicity of infection (MOI) of 0.1, were purified through a 25% w/v sucrose followed by a 25–65% sucrose gradient. Purified BVs were resuspended in PBS, and nucleocapsids and envelopes were separated using 1% IGEPAL and ultracentrifugation through a glycerol cushion. Occlusion derived viruses (ODVs) were obtained after alkaline treatment of polyhedra recovered from the infected Sf-9 cells. ODVs were purified through a discontinuous 30–65% sucrose gradient. Ac109, GP64 and VP39 proteins were detected from purified ODVs, BVs, BVs envelopes (Env) and nucleocapsid (Nc) fractions with a polyclonal serum anti Ac109-GST fusion protein, an anti-GP64 monoclonal antibody or an anti-VP39 monoclonal antibody. Sf-9 cells infected with wt Ac MNPV (IC) were used as a control of Ac109 detection. (B) Subcellular localization of Ac109 in living cells. Ac109 fused to YFP protein at either C or N-terminus (Ac109:YFP and YFP:Ac109) and transcribed from pOpIE2 promoter was detected in Sf-9.RNuc cells by confocal microscopy at 48 h post-transfection (hpt). (C) Kinetics of subcellular localization of Ac109 during baculovirus infection. Confocal microscopy of Sf-9.RNuc cells infected with Acppolp109-109Y virus at a MOI of 5 is shown at indicated h post-infection (hpi). Ac109 was fused to YFP at the C-terminal end (Ac109:YFP) and transcribed from the ac109 promoter. (D) Living insect cells infected with Acppolp109-109Y virus showing a heterogeneous Ac109:YFP accumulation around forming polyhedra in the nucleus. (E) Ac109:YFP fluorescence concentrated in the already formed polyhedra. (F) Cytoplasmic accumulations of Ac109:YFP in Sf-9.RNuc cells at different stages of viral infection. Bars represent 10 µm.
    Figure Legend Snippet: Ac109 localization in virions and in Sf-9 cells. (A) Western blot analysis of purified virions. Budded viruses (BVs), obtained from supernatants of Sf-9 cells infected with wild type (wt) Ac MNPV at a multiplicity of infection (MOI) of 0.1, were purified through a 25% w/v sucrose followed by a 25–65% sucrose gradient. Purified BVs were resuspended in PBS, and nucleocapsids and envelopes were separated using 1% IGEPAL and ultracentrifugation through a glycerol cushion. Occlusion derived viruses (ODVs) were obtained after alkaline treatment of polyhedra recovered from the infected Sf-9 cells. ODVs were purified through a discontinuous 30–65% sucrose gradient. Ac109, GP64 and VP39 proteins were detected from purified ODVs, BVs, BVs envelopes (Env) and nucleocapsid (Nc) fractions with a polyclonal serum anti Ac109-GST fusion protein, an anti-GP64 monoclonal antibody or an anti-VP39 monoclonal antibody. Sf-9 cells infected with wt Ac MNPV (IC) were used as a control of Ac109 detection. (B) Subcellular localization of Ac109 in living cells. Ac109 fused to YFP protein at either C or N-terminus (Ac109:YFP and YFP:Ac109) and transcribed from pOpIE2 promoter was detected in Sf-9.RNuc cells by confocal microscopy at 48 h post-transfection (hpt). (C) Kinetics of subcellular localization of Ac109 during baculovirus infection. Confocal microscopy of Sf-9.RNuc cells infected with Acppolp109-109Y virus at a MOI of 5 is shown at indicated h post-infection (hpi). Ac109 was fused to YFP at the C-terminal end (Ac109:YFP) and transcribed from the ac109 promoter. (D) Living insect cells infected with Acppolp109-109Y virus showing a heterogeneous Ac109:YFP accumulation around forming polyhedra in the nucleus. (E) Ac109:YFP fluorescence concentrated in the already formed polyhedra. (F) Cytoplasmic accumulations of Ac109:YFP in Sf-9.RNuc cells at different stages of viral infection. Bars represent 10 µm.

    Techniques Used: Western Blot, Purification, Infection, Derivative Assay, Confocal Microscopy, Transfection, Fluorescence

    2) Product Images from "Protein Translocation by Bacterial Toxin Channels: A Comparison of Diphtheria Toxin and Colicin Ia"

    Article Title: Protein Translocation by Bacterial Toxin Channels: A Comparison of Diphtheria Toxin and Colicin Ia

    Journal:

    doi: 10.1529/biophysj.106.085753

    Linear sequence of the A-Ia construct used for these experiments. The 190 residues of the A chain of DTA are preceded by the His 6 -tag and thrombin recognition sequences from pET15-b and connected to the channel-forming domain (residues 438–626)
    Figure Legend Snippet: Linear sequence of the A-Ia construct used for these experiments. The 190 residues of the A chain of DTA are preceded by the His 6 -tag and thrombin recognition sequences from pET15-b and connected to the channel-forming domain (residues 438–626)

    Techniques Used: Sequencing, IA, Construct

    3) Product Images from "Biochemical and Structural Properties of Cyanases from Arabidopsis thaliana and Oryza sativa"

    Article Title: Biochemical and Structural Properties of Cyanases from Arabidopsis thaliana and Oryza sativa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018300

    Identification of Atcyn mutant plants and transgenic plants. (A) Schematic diagram of the AtCYN gene and the T-DNA insertion positions. Gray boxes represent exons. Lines above the gene indicate T-DNA insertion positions. Accession numbers of the Atcyn mutant lines was listed in Table 4 . (B) Northern blot analysis of AtCYN transcripts in cyn mutants. Total RNA was isolated from 14-day-old seedlings. Different homozygous individuals identified were analyzed in different lanes. Blot signals (indicated by the arrow) were quantified with ImageJ version 1.4 software and the values are presented below each lane. The 5S rRNA was visualized with ultraviolet light and was the loading control. (C) Quantitative RT-PCR analysis of AtCYN transcripts in Col 0, cyn7 and transgenic plants. Error bars represent the standard deviation of three biological replicates. (D) Semi-quantitative analysis of HA:AtCYN and HA:OsCYN in transgenic plants using western blotting (WB). Blot signals (indicated by the arrow) were quantified with ImageJ and the values are presented below the lanes. The large subunit of Rubisco was visualised by Coomassie Brilliant Blue (CBB) and was the total protein loading control (indicated by the arrow).
    Figure Legend Snippet: Identification of Atcyn mutant plants and transgenic plants. (A) Schematic diagram of the AtCYN gene and the T-DNA insertion positions. Gray boxes represent exons. Lines above the gene indicate T-DNA insertion positions. Accession numbers of the Atcyn mutant lines was listed in Table 4 . (B) Northern blot analysis of AtCYN transcripts in cyn mutants. Total RNA was isolated from 14-day-old seedlings. Different homozygous individuals identified were analyzed in different lanes. Blot signals (indicated by the arrow) were quantified with ImageJ version 1.4 software and the values are presented below each lane. The 5S rRNA was visualized with ultraviolet light and was the loading control. (C) Quantitative RT-PCR analysis of AtCYN transcripts in Col 0, cyn7 and transgenic plants. Error bars represent the standard deviation of three biological replicates. (D) Semi-quantitative analysis of HA:AtCYN and HA:OsCYN in transgenic plants using western blotting (WB). Blot signals (indicated by the arrow) were quantified with ImageJ and the values are presented below the lanes. The large subunit of Rubisco was visualised by Coomassie Brilliant Blue (CBB) and was the total protein loading control (indicated by the arrow).

    Techniques Used: Mutagenesis, Transgenic Assay, Northern Blot, Isolation, Software, Quantitative RT-PCR, Standard Deviation, Western Blot

    Influence of pH and temperature on cyanase activity. The His:AtCYN and His:OsCYN enzymes were purified and their activities assayed in vitro . (A) Effect of pH on cyanase activity at 27°C. BSA was used as a control. (B) Effect of temperature on cyanase activity at pH 7.7.
    Figure Legend Snippet: Influence of pH and temperature on cyanase activity. The His:AtCYN and His:OsCYN enzymes were purified and their activities assayed in vitro . (A) Effect of pH on cyanase activity at 27°C. BSA was used as a control. (B) Effect of temperature on cyanase activity at pH 7.7.

    Techniques Used: Activity Assay, Purification, In Vitro

    Gel filtration and cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A). (A) Gel filtration. High Molecular Weight (HMW) Standard: Thyroglobulin, 669 kDa; Ferritin, 440 kDa; Aldolase, 158 kDa; Conalbumin, 75 kDa and Ovalbumin, 43 kDa. And calculated molecular weight: AtCYN, 210.74 kDa; OsCYN, 211.72 kDa; AtCYN-E94L, 71.64 kDa and AtCYN-S117A, 62.76 kDa. (B) Cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A) at pH 7.7 and 27°C.
    Figure Legend Snippet: Gel filtration and cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A). (A) Gel filtration. High Molecular Weight (HMW) Standard: Thyroglobulin, 669 kDa; Ferritin, 440 kDa; Aldolase, 158 kDa; Conalbumin, 75 kDa and Ovalbumin, 43 kDa. And calculated molecular weight: AtCYN, 210.74 kDa; OsCYN, 211.72 kDa; AtCYN-E94L, 71.64 kDa and AtCYN-S117A, 62.76 kDa. (B) Cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A) at pH 7.7 and 27°C.

    Techniques Used: Filtration, Molecular Weight

    Homology modelling of AtCYN and OsCYN. (A) The predicted structures of AtCYN (blue) and OsCYN (magentas) were similar to the crystal structure of the EcCYN monomer (green). Ball-and-stick figures represent the conserved catalytic residues Arg96, Glu99 (B) and Ser122 of the EcCYN (C). Red dots indicate chloride ions.
    Figure Legend Snippet: Homology modelling of AtCYN and OsCYN. (A) The predicted structures of AtCYN (blue) and OsCYN (magentas) were similar to the crystal structure of the EcCYN monomer (green). Ball-and-stick figures represent the conserved catalytic residues Arg96, Glu99 (B) and Ser122 of the EcCYN (C). Red dots indicate chloride ions.

    Techniques Used:

    Decomposition of cyanate by AtCYN and OsCYN in vivo . Seeds were plated on MS medium containing either 0 mM, 0.5 mM, 1 mM or 2 mM KCNO. Plates were incubated at 4°C for 3 days and then transferred to a growth chamber for 7 days.
    Figure Legend Snippet: Decomposition of cyanate by AtCYN and OsCYN in vivo . Seeds were plated on MS medium containing either 0 mM, 0.5 mM, 1 mM or 2 mM KCNO. Plates were incubated at 4°C for 3 days and then transferred to a growth chamber for 7 days.

    Techniques Used: In Vivo, Mass Spectrometry, Incubation

    Coimmunoprecipitation assay demonstrating self-interaction of AtCYN (A) and OsCYN (B). Although an extremely low amount of OsCYN proteins was detected in the input samples, an identical pattern was shown by OsCYN in the assay. Lane 1: Mock; Lane 2: HA:CYN and FLAG:CYN were detected in the input samples; Lane 3: when anti-HA antibody was added, FLAG:CYN immunoprecipitated with HA:CYN (Lanes 6 8 were controls); Lane 4: when anti-FLAG antibody was added, HA:CYN immunoprecipitated with FLAG:CYN (Lanes 7 9 were controls); Lane 5: Native mouse IgG was used as negative control of antibodies. Bands of HA:CYN and Flag:CYN are indicated with arrows and bands of mouse IgG are indicated with stars.
    Figure Legend Snippet: Coimmunoprecipitation assay demonstrating self-interaction of AtCYN (A) and OsCYN (B). Although an extremely low amount of OsCYN proteins was detected in the input samples, an identical pattern was shown by OsCYN in the assay. Lane 1: Mock; Lane 2: HA:CYN and FLAG:CYN were detected in the input samples; Lane 3: when anti-HA antibody was added, FLAG:CYN immunoprecipitated with HA:CYN (Lanes 6 8 were controls); Lane 4: when anti-FLAG antibody was added, HA:CYN immunoprecipitated with FLAG:CYN (Lanes 7 9 were controls); Lane 5: Native mouse IgG was used as negative control of antibodies. Bands of HA:CYN and Flag:CYN are indicated with arrows and bands of mouse IgG are indicated with stars.

    Techniques Used: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control

    4) Product Images from "Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila"

    Article Title: Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.187

    Purification and crosslinking of complexes formed between AcrH and AopB or AopD. A: HiLoad 16/60 Superdex 200 elution profile of AcrH and SDS-PAGE showing crosslinking of AcrH. Lane (1) M w marker and lane (2) crosslinked product of AcrH. The major product
    Figure Legend Snippet: Purification and crosslinking of complexes formed between AcrH and AopB or AopD. A: HiLoad 16/60 Superdex 200 elution profile of AcrH and SDS-PAGE showing crosslinking of AcrH. Lane (1) M w marker and lane (2) crosslinked product of AcrH. The major product

    Techniques Used: Purification, SDS Page, Marker

    Limited protease digestion of the AcrH-AopB complex. A: SDS-PAGE showing partial digestion of AcrH-AopB with elastase. Lane (1) M w marker; lane (2) monomeric AcrH-AopB from gel filtration before digestion; and lane (3) AopB is being digested into a single
    Figure Legend Snippet: Limited protease digestion of the AcrH-AopB complex. A: SDS-PAGE showing partial digestion of AcrH-AopB with elastase. Lane (1) M w marker; lane (2) monomeric AcrH-AopB from gel filtration before digestion; and lane (3) AopB is being digested into a single

    Techniques Used: SDS Page, Marker, Filtration

    Limited protease digestion of the AcrH-AopD complex. A: SDS-PAGE showing partial digestion of AcrH-AopD with chymotrypsin. Lane (1) M w marker; lane (2) monomeric AcrH-AopD from gel filtration before digestion; and lane (3) AopD is being digested into
    Figure Legend Snippet: Limited protease digestion of the AcrH-AopD complex. A: SDS-PAGE showing partial digestion of AcrH-AopD with chymotrypsin. Lane (1) M w marker; lane (2) monomeric AcrH-AopD from gel filtration before digestion; and lane (3) AopD is being digested into

    Techniques Used: SDS Page, Marker, Filtration

    Thermal denaturation of AcrH and various complexes as monitored by FarUV-CD at 222 nm. The fraction of protein denatured is plotted against temperature (°C). Legends for different proteins are: dimeric AcrH (open circle); monomeric AcrH-AopB (open
    Figure Legend Snippet: Thermal denaturation of AcrH and various complexes as monitored by FarUV-CD at 222 nm. The fraction of protein denatured is plotted against temperature (°C). Legends for different proteins are: dimeric AcrH (open circle); monomeric AcrH-AopB (open

    Techniques Used:

    Purification of the AcrH-AopB-AopD and the AcrH-AopB 1–264 -AopD complexes. A: Purification of the AcrH-AopB-AopD complex. Lane (1) M w marker and lane (2) purified AcrH-AopB-AopD complex from Ni-NTA column with His-tag placed on AopD. The gel filtration
    Figure Legend Snippet: Purification of the AcrH-AopB-AopD and the AcrH-AopB 1–264 -AopD complexes. A: Purification of the AcrH-AopB-AopD complex. Lane (1) M w marker and lane (2) purified AcrH-AopB-AopD complex from Ni-NTA column with His-tag placed on AopD. The gel filtration

    Techniques Used: Purification, Marker, Filtration

    5) Product Images from "Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host"

    Article Title: Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host

    Journal: Journal of Immunology Research

    doi: 10.1155/2019/6793596

    Purification of recombinant Sm16. (a) Amino acid sequence of Schistosoma mansoni Sm16 (GenBank: AAD26122.1 and WormBase ParaSite: Smp_341790). The signal peptide is underlined and the sequence in bold corresponds to amino acids 23 to 90. (b) Synthetic gene construction containing the DNA sequence corresponding to the region encoding the amino acids 23 to 90 with restriction enzyme sites BamH I and Xho I at the 5′ and 3′ ends, respectively, and the initiation codon ATG in bold. (c) 15% SDS-PAGE of purified rSm16 stained by Coomassie Blue R-250 and Western blot using monoclonal 6x-His-tag antibody. Molecular weight markers Dual Color (Bio-Rad) are indicated in kDa.
    Figure Legend Snippet: Purification of recombinant Sm16. (a) Amino acid sequence of Schistosoma mansoni Sm16 (GenBank: AAD26122.1 and WormBase ParaSite: Smp_341790). The signal peptide is underlined and the sequence in bold corresponds to amino acids 23 to 90. (b) Synthetic gene construction containing the DNA sequence corresponding to the region encoding the amino acids 23 to 90 with restriction enzyme sites BamH I and Xho I at the 5′ and 3′ ends, respectively, and the initiation codon ATG in bold. (c) 15% SDS-PAGE of purified rSm16 stained by Coomassie Blue R-250 and Western blot using monoclonal 6x-His-tag antibody. Molecular weight markers Dual Color (Bio-Rad) are indicated in kDa.

    Techniques Used: Purification, Recombinant, Sequencing, SDS Page, Staining, Western Blot, Molecular Weight

    6) Product Images from "Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis"

    Article Title: Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013143

    Purification of the Rv0045c protein by ion exchange chromatography and gel filtration chromatography. The Rv0045c protein was purified by anion exchange chromatography (A), cation exchange chromatography (B), and gel filtration chromatography (C). The purity was checked by SDS-PAGE analysis after each purification procedure.
    Figure Legend Snippet: Purification of the Rv0045c protein by ion exchange chromatography and gel filtration chromatography. The Rv0045c protein was purified by anion exchange chromatography (A), cation exchange chromatography (B), and gel filtration chromatography (C). The purity was checked by SDS-PAGE analysis after each purification procedure.

    Techniques Used: Purification, Ion Exchange Chromatography, Filtration, Chromatography, SDS Page

    MALDI-TOF peptide mass fingerprint (PMF) spectrometry of the Rv0045c protein. The PMF analysis was made from fragments of purified Rv0045c protein derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments was shown in bold red.
    Figure Legend Snippet: MALDI-TOF peptide mass fingerprint (PMF) spectrometry of the Rv0045c protein. The PMF analysis was made from fragments of purified Rv0045c protein derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments was shown in bold red.

    Techniques Used: Peptide Mass Fingerprinting, Purification, Derivative Assay, Sequencing

    CD spectra of the Rv0045c protein at different pH and temperatures. The CD measurements were made in the presence of various pH (A) at pH 2.0 (black), pH 3.0 (red), pH 4.0 (yellow), pH 6.0 (blue), pH 7.0 (purple), pH 8.0 (pink), pH 9.0 (green), pH 10.0 (gray), pH 11.0 (coral) and pH 12.0 (light green) at room temperature, and different temperatures (B) at 10°C (black), 20°C (gray), 30°C (yellow), 40°C (green), 50°C (blue), 60°C (purple) and 70°C (red) at pH 7.5, respectively. Values represent the mean ± SD of three analyses. The concentration of the Rv0045c protein was fixed at 0.35 mg/mL (20 mM Tris, pH 7.5).
    Figure Legend Snippet: CD spectra of the Rv0045c protein at different pH and temperatures. The CD measurements were made in the presence of various pH (A) at pH 2.0 (black), pH 3.0 (red), pH 4.0 (yellow), pH 6.0 (blue), pH 7.0 (purple), pH 8.0 (pink), pH 9.0 (green), pH 10.0 (gray), pH 11.0 (coral) and pH 12.0 (light green) at room temperature, and different temperatures (B) at 10°C (black), 20°C (gray), 30°C (yellow), 40°C (green), 50°C (blue), 60°C (purple) and 70°C (red) at pH 7.5, respectively. Values represent the mean ± SD of three analyses. The concentration of the Rv0045c protein was fixed at 0.35 mg/mL (20 mM Tris, pH 7.5).

    Techniques Used: Concentration Assay

    Effects of temperature and pH on enzyme activity of the Rv0045c protein. The enzyme activities were measured using p-butyrate caprylate (C 6 ) as substrate in the presence of mild temperatures (36°C–40°C) at pH 6.0 (red), pH 7.0 (green) and pH 8.0 (blue). Values represent the mean ± SD of five analyses. The concentration of the Rvoo45c protein was fixed at 0.2 mg/mL (20 mM Tris, pH 7.5). The enzyme activities were expressed as units hydrolase/mg protein/min (one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min).
    Figure Legend Snippet: Effects of temperature and pH on enzyme activity of the Rv0045c protein. The enzyme activities were measured using p-butyrate caprylate (C 6 ) as substrate in the presence of mild temperatures (36°C–40°C) at pH 6.0 (red), pH 7.0 (green) and pH 8.0 (blue). Values represent the mean ± SD of five analyses. The concentration of the Rvoo45c protein was fixed at 0.2 mg/mL (20 mM Tris, pH 7.5). The enzyme activities were expressed as units hydrolase/mg protein/min (one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min).

    Techniques Used: Activity Assay, Concentration Assay

    SDS-PAGE analysis for expression and affinity chromatography of the Rv0045c protein. Lane 1, culture pellet (uninduced); Lane 2, culture pellet (induced with 0.3 mM IPTG at 16°C); Lane 3, the supernatant of induced cells after sonication; Lane 4, fluid through Ni 2+ -affinity chromatography column; Lane 5 and 7: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 200 mM Imidazole, pH 7.5; Lane 6 and 8: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 500 mM Imidazole, pH 7.5; Lane M: molecular mass markers.
    Figure Legend Snippet: SDS-PAGE analysis for expression and affinity chromatography of the Rv0045c protein. Lane 1, culture pellet (uninduced); Lane 2, culture pellet (induced with 0.3 mM IPTG at 16°C); Lane 3, the supernatant of induced cells after sonication; Lane 4, fluid through Ni 2+ -affinity chromatography column; Lane 5 and 7: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 200 mM Imidazole, pH 7.5; Lane 6 and 8: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 500 mM Imidazole, pH 7.5; Lane M: molecular mass markers.

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

    7) Product Images from "Sublingual Immunization with M2-Based Vaccine Induces Broad Protective Immunity against Influenza"

    Article Title: Sublingual Immunization with M2-Based Vaccine Induces Broad Protective Immunity against Influenza

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027953

    3M2eC-specific Ab levels in secretions and lung tissues. Mice were immunized with 10 ug of 3M2eC protein plus 2 ug of CT via i.n. or s.l., or with 10 ug of 3M2eC protein plus alum via i.d. or i.m. on day 0, 14, and 28. Saliva, nasal wash and BAL were collected two weeks after last immunization. M2e-specific IgA in the secretions (A) and M2e-specific IgG in BAL (B) were determined by ELISA using 3M2eC protein. (C) Number of M2e-specific IgG or IgA Ab secreting cells in the lung tissue at day 7 after last immunization was determined by ELISPOT using 3M2eC protein. N.D., not detected. The dashed line shows the limit of detection. The results are expressed as the means+S.D. for the group (n = 5). The data are representative of three independent experiments.
    Figure Legend Snippet: 3M2eC-specific Ab levels in secretions and lung tissues. Mice were immunized with 10 ug of 3M2eC protein plus 2 ug of CT via i.n. or s.l., or with 10 ug of 3M2eC protein plus alum via i.d. or i.m. on day 0, 14, and 28. Saliva, nasal wash and BAL were collected two weeks after last immunization. M2e-specific IgA in the secretions (A) and M2e-specific IgG in BAL (B) were determined by ELISA using 3M2eC protein. (C) Number of M2e-specific IgG or IgA Ab secreting cells in the lung tissue at day 7 after last immunization was determined by ELISPOT using 3M2eC protein. N.D., not detected. The dashed line shows the limit of detection. The results are expressed as the means+S.D. for the group (n = 5). The data are representative of three independent experiments.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

    Construction of plasmids and purification of M2 proteins. (A) The synthetic M2eC or 3M2eC genes without hydrophobic region (amino acids 26–55) from PR8 virus were cloned into pET15b vector (B). The recombinant proteins expressed in E. coli were purified by His-tag affinity chromatography and detected by Western blot using M2e-specific monoclonal Ab, 14C2.
    Figure Legend Snippet: Construction of plasmids and purification of M2 proteins. (A) The synthetic M2eC or 3M2eC genes without hydrophobic region (amino acids 26–55) from PR8 virus were cloned into pET15b vector (B). The recombinant proteins expressed in E. coli were purified by His-tag affinity chromatography and detected by Western blot using M2e-specific monoclonal Ab, 14C2.

    Techniques Used: Purification, Clone Assay, Plasmid Preparation, Recombinant, Affinity Chromatography, Western Blot

    Immunogenicity of 3M2eC (A B): BALB/c mice were immunized i.n. with 10 ug of M2eC, 3M2eC, or 3M2eC plus 2 ug of CT on day 0 and 14. Mice received PBS serve as control group. Sera and saliva were collected on day 14 after last immunization. Levels of M2e-specific IgG in sera (A) and IgA in saliva (B) were determined by ELISA. Ab levels induced by different immunization methods (C D): BALB/c mice were administered on day 0 and 14 with 10 ug of 3M2eC protein plus 2 ug of CT for i.n. and s.l. immunizations or plus alum i.d. or i.m. immunizations. Sera were collected on day 14 after the last immunization. Ab and analyzed for M2eC-specific IgG subclasses by ELISA using 3M2eC protein (C) and M2e-specific IgG Ab by ELISA using M2e-expressing Hela cells (D). N.D., not detected. The dashed line shows the limit of detection. The results are expressed as the means+S.D. for the group (n = 5). The data are representative of three independent experiments. Significant differences were expressed as *, P
    Figure Legend Snippet: Immunogenicity of 3M2eC (A B): BALB/c mice were immunized i.n. with 10 ug of M2eC, 3M2eC, or 3M2eC plus 2 ug of CT on day 0 and 14. Mice received PBS serve as control group. Sera and saliva were collected on day 14 after last immunization. Levels of M2e-specific IgG in sera (A) and IgA in saliva (B) were determined by ELISA. Ab levels induced by different immunization methods (C D): BALB/c mice were administered on day 0 and 14 with 10 ug of 3M2eC protein plus 2 ug of CT for i.n. and s.l. immunizations or plus alum i.d. or i.m. immunizations. Sera were collected on day 14 after the last immunization. Ab and analyzed for M2eC-specific IgG subclasses by ELISA using 3M2eC protein (C) and M2e-specific IgG Ab by ELISA using M2e-expressing Hela cells (D). N.D., not detected. The dashed line shows the limit of detection. The results are expressed as the means+S.D. for the group (n = 5). The data are representative of three independent experiments. Significant differences were expressed as *, P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Expressing

    Protection against the 2009 pandemic influenza A virus (H1N1). Mice were immunized i.n. or s.l. with 3M2eC (10 ug) plus CT (2 ug) on days 0 and 14 and challenged by i.n. administration of A/CA/04/09 (H1N1) 5 weeks after the last immunization. (A) Virus titers in the lung tissue at day 5 after challenge were determined in embryonated chicken eggs. (B) Body weight was monitored daily after the viral challenge. The results are expressed as the means+S.D. for the group. Significant differences were expressed as *, P
    Figure Legend Snippet: Protection against the 2009 pandemic influenza A virus (H1N1). Mice were immunized i.n. or s.l. with 3M2eC (10 ug) plus CT (2 ug) on days 0 and 14 and challenged by i.n. administration of A/CA/04/09 (H1N1) 5 weeks after the last immunization. (A) Virus titers in the lung tissue at day 5 after challenge were determined in embryonated chicken eggs. (B) Body weight was monitored daily after the viral challenge. The results are expressed as the means+S.D. for the group. Significant differences were expressed as *, P

    Techniques Used: Mouse Assay

    Cross-protection against infections with different influenza virus subtypes. Six-week-old female BALB/c mice (n = 6) were immunized twice with 10 ug of 3M2eC protein plus 2 ug of CT at 2 week intervals via i.n. or s.l., or with 10 ug of 3M2eC protein plus alum by i.d. or i.m.. They were challenged i.n. with 10 LD 50 of mouse adapted PR8 strain (H1N1) at 3 weeks (A and B), A/Aquatic Bird/Korea/W81/05 virus (H5N2) at 3 weeks (C and D) or A/Philippine/2/82 (H3N2) virus at 5 weeks (E and F) after the last immunization. Survival rate and the body weight loss were monitored daily after the challenge. The results are expressed as the means+S.D. for the group.
    Figure Legend Snippet: Cross-protection against infections with different influenza virus subtypes. Six-week-old female BALB/c mice (n = 6) were immunized twice with 10 ug of 3M2eC protein plus 2 ug of CT at 2 week intervals via i.n. or s.l., or with 10 ug of 3M2eC protein plus alum by i.d. or i.m.. They were challenged i.n. with 10 LD 50 of mouse adapted PR8 strain (H1N1) at 3 weeks (A and B), A/Aquatic Bird/Korea/W81/05 virus (H5N2) at 3 weeks (C and D) or A/Philippine/2/82 (H3N2) virus at 5 weeks (E and F) after the last immunization. Survival rate and the body weight loss were monitored daily after the challenge. The results are expressed as the means+S.D. for the group.

    Techniques Used: Mouse Assay

    8) Product Images from "Characterization of the Entamoeba histolytica Ornithine Decarboxylase-Like Enzyme"

    Article Title: Characterization of the Entamoeba histolytica Ornithine Decarboxylase-Like Enzyme

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0000115

    Overexpression and purification of E. histolytica putative ornithine decarboxylase-like protein. (A) Coomassie blue staining of SDS/PAGE showing overexpression of full-length E. histolytica putative ODC-like protein in E. coli. The pET 30a bacterial extract after induction (lanes 1 and 2) at 3 h and 1 h, respectively with 1mM IPTG and before induction (lane 3). The arrow shows the induced recombinant putative ODC-like protein. The broad-range protein MW marker (lane 4) (BioRad) was used to identify the size of recombinant protein. (B) Purification of putative ODC-likeprotein on Ni 2+ affinity resin. Lanes 1–4, eluted fractions showing purified putative ODC-like protein from affinity column; lanes 5, 6, and 8 are washes; lane 7, broad-range protein MW marker (BioRad); lane 9, supernatant from the crude lysate. (C) Western blot using anti-His antibody. Western blot analysis of different concentrations of purified putative ODC-His fusion recombinant protein. Lanes 1–5 represent 25, 20, 18, 15, and 5 µg of recombinant proteins, respectively. (D) Western blot using anti- E. histolytica ODC. Lanes 1–3 represent 2, 3, and 6 µg of purified recombinant protein. Prestained broad-range protein molecular weight marker (BioRad) was used to identify the size of the protein on the Western blot. (E) Western blot using anti- E. histolytica ODC. Lane 1, purified recombinant protein. Lane 2, E. histolytica lysate. Prestained broad range protein molecular weight marker (BioRad) was used to identify the size of the protein on the Western blot.
    Figure Legend Snippet: Overexpression and purification of E. histolytica putative ornithine decarboxylase-like protein. (A) Coomassie blue staining of SDS/PAGE showing overexpression of full-length E. histolytica putative ODC-like protein in E. coli. The pET 30a bacterial extract after induction (lanes 1 and 2) at 3 h and 1 h, respectively with 1mM IPTG and before induction (lane 3). The arrow shows the induced recombinant putative ODC-like protein. The broad-range protein MW marker (lane 4) (BioRad) was used to identify the size of recombinant protein. (B) Purification of putative ODC-likeprotein on Ni 2+ affinity resin. Lanes 1–4, eluted fractions showing purified putative ODC-like protein from affinity column; lanes 5, 6, and 8 are washes; lane 7, broad-range protein MW marker (BioRad); lane 9, supernatant from the crude lysate. (C) Western blot using anti-His antibody. Western blot analysis of different concentrations of purified putative ODC-His fusion recombinant protein. Lanes 1–5 represent 25, 20, 18, 15, and 5 µg of recombinant proteins, respectively. (D) Western blot using anti- E. histolytica ODC. Lanes 1–3 represent 2, 3, and 6 µg of purified recombinant protein. Prestained broad-range protein molecular weight marker (BioRad) was used to identify the size of the protein on the Western blot. (E) Western blot using anti- E. histolytica ODC. Lane 1, purified recombinant protein. Lane 2, E. histolytica lysate. Prestained broad range protein molecular weight marker (BioRad) was used to identify the size of the protein on the Western blot.

    Techniques Used: Over Expression, Purification, Staining, SDS Page, Positron Emission Tomography, Recombinant, Marker, Affinity Column, Western Blot, Molecular Weight

    9) Product Images from "Biochemical and Structural Properties of Cyanases from Arabidopsis thaliana and Oryza sativa"

    Article Title: Biochemical and Structural Properties of Cyanases from Arabidopsis thaliana and Oryza sativa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018300

    Identification of Atcyn mutant plants and transgenic plants. (A) Schematic diagram of the AtCYN gene and the T-DNA insertion positions. Gray boxes represent exons. Lines above the gene indicate T-DNA insertion positions. Accession numbers of the Atcyn mutant lines was listed in Table 4 . (B) Northern blot analysis of AtCYN transcripts in cyn mutants. Total RNA was isolated from 14-day-old seedlings. Different homozygous individuals identified were analyzed in different lanes. Blot signals (indicated by the arrow) were quantified with ImageJ version 1.4 software and the values are presented below each lane. The 5S rRNA was visualized with ultraviolet light and was the loading control. (C) Quantitative RT-PCR analysis of AtCYN transcripts in Col 0, cyn7 and transgenic plants. Error bars represent the standard deviation of three biological replicates. (D) Semi-quantitative analysis of HA:AtCYN and HA:OsCYN in transgenic plants using western blotting (WB). Blot signals (indicated by the arrow) were quantified with ImageJ and the values are presented below the lanes. The large subunit of Rubisco was visualised by Coomassie Brilliant Blue (CBB) and was the total protein loading control (indicated by the arrow).
    Figure Legend Snippet: Identification of Atcyn mutant plants and transgenic plants. (A) Schematic diagram of the AtCYN gene and the T-DNA insertion positions. Gray boxes represent exons. Lines above the gene indicate T-DNA insertion positions. Accession numbers of the Atcyn mutant lines was listed in Table 4 . (B) Northern blot analysis of AtCYN transcripts in cyn mutants. Total RNA was isolated from 14-day-old seedlings. Different homozygous individuals identified were analyzed in different lanes. Blot signals (indicated by the arrow) were quantified with ImageJ version 1.4 software and the values are presented below each lane. The 5S rRNA was visualized with ultraviolet light and was the loading control. (C) Quantitative RT-PCR analysis of AtCYN transcripts in Col 0, cyn7 and transgenic plants. Error bars represent the standard deviation of three biological replicates. (D) Semi-quantitative analysis of HA:AtCYN and HA:OsCYN in transgenic plants using western blotting (WB). Blot signals (indicated by the arrow) were quantified with ImageJ and the values are presented below the lanes. The large subunit of Rubisco was visualised by Coomassie Brilliant Blue (CBB) and was the total protein loading control (indicated by the arrow).

    Techniques Used: Mutagenesis, Transgenic Assay, Northern Blot, Isolation, Software, Quantitative RT-PCR, Standard Deviation, Western Blot

    Influence of pH and temperature on cyanase activity. The His:AtCYN and His:OsCYN enzymes were purified and their activities assayed in vitro . (A) Effect of pH on cyanase activity at 27°C. BSA was used as a control. (B) Effect of temperature on cyanase activity at pH 7.7.
    Figure Legend Snippet: Influence of pH and temperature on cyanase activity. The His:AtCYN and His:OsCYN enzymes were purified and their activities assayed in vitro . (A) Effect of pH on cyanase activity at 27°C. BSA was used as a control. (B) Effect of temperature on cyanase activity at pH 7.7.

    Techniques Used: Activity Assay, Purification, In Vitro

    Gel filtration and cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A). (A) Gel filtration. High Molecular Weight (HMW) Standard: Thyroglobulin, 669 kDa; Ferritin, 440 kDa; Aldolase, 158 kDa; Conalbumin, 75 kDa and Ovalbumin, 43 kDa. And calculated molecular weight: AtCYN, 210.74 kDa; OsCYN, 211.72 kDa; AtCYN-E94L, 71.64 kDa and AtCYN-S117A, 62.76 kDa. (B) Cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A) at pH 7.7 and 27°C.
    Figure Legend Snippet: Gel filtration and cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A). (A) Gel filtration. High Molecular Weight (HMW) Standard: Thyroglobulin, 669 kDa; Ferritin, 440 kDa; Aldolase, 158 kDa; Conalbumin, 75 kDa and Ovalbumin, 43 kDa. And calculated molecular weight: AtCYN, 210.74 kDa; OsCYN, 211.72 kDa; AtCYN-E94L, 71.64 kDa and AtCYN-S117A, 62.76 kDa. (B) Cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A) at pH 7.7 and 27°C.

    Techniques Used: Filtration, Molecular Weight

    Homology modelling of AtCYN and OsCYN. (A) The predicted structures of AtCYN (blue) and OsCYN (magentas) were similar to the crystal structure of the EcCYN monomer (green). Ball-and-stick figures represent the conserved catalytic residues Arg96, Glu99 (B) and Ser122 of the EcCYN (C). Red dots indicate chloride ions.
    Figure Legend Snippet: Homology modelling of AtCYN and OsCYN. (A) The predicted structures of AtCYN (blue) and OsCYN (magentas) were similar to the crystal structure of the EcCYN monomer (green). Ball-and-stick figures represent the conserved catalytic residues Arg96, Glu99 (B) and Ser122 of the EcCYN (C). Red dots indicate chloride ions.

    Techniques Used:

    Decomposition of cyanate by AtCYN and OsCYN in vivo . Seeds were plated on MS medium containing either 0 mM, 0.5 mM, 1 mM or 2 mM KCNO. Plates were incubated at 4°C for 3 days and then transferred to a growth chamber for 7 days.
    Figure Legend Snippet: Decomposition of cyanate by AtCYN and OsCYN in vivo . Seeds were plated on MS medium containing either 0 mM, 0.5 mM, 1 mM or 2 mM KCNO. Plates were incubated at 4°C for 3 days and then transferred to a growth chamber for 7 days.

    Techniques Used: In Vivo, Mass Spectrometry, Incubation

    Coimmunoprecipitation assay demonstrating self-interaction of AtCYN (A) and OsCYN (B). Although an extremely low amount of OsCYN proteins was detected in the input samples, an identical pattern was shown by OsCYN in the assay. Lane 1: Mock; Lane 2: HA:CYN and FLAG:CYN were detected in the input samples; Lane 3: when anti-HA antibody was added, FLAG:CYN immunoprecipitated with HA:CYN (Lanes 6 8 were controls); Lane 4: when anti-FLAG antibody was added, HA:CYN immunoprecipitated with FLAG:CYN (Lanes 7 9 were controls); Lane 5: Native mouse IgG was used as negative control of antibodies. Bands of HA:CYN and Flag:CYN are indicated with arrows and bands of mouse IgG are indicated with stars.
    Figure Legend Snippet: Coimmunoprecipitation assay demonstrating self-interaction of AtCYN (A) and OsCYN (B). Although an extremely low amount of OsCYN proteins was detected in the input samples, an identical pattern was shown by OsCYN in the assay. Lane 1: Mock; Lane 2: HA:CYN and FLAG:CYN were detected in the input samples; Lane 3: when anti-HA antibody was added, FLAG:CYN immunoprecipitated with HA:CYN (Lanes 6 8 were controls); Lane 4: when anti-FLAG antibody was added, HA:CYN immunoprecipitated with FLAG:CYN (Lanes 7 9 were controls); Lane 5: Native mouse IgG was used as negative control of antibodies. Bands of HA:CYN and Flag:CYN are indicated with arrows and bands of mouse IgG are indicated with stars.

    Techniques Used: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control

    10) Product Images from "Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila"

    Article Title: Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.187

    Purification and crosslinking of complexes formed between AcrH and AopB or AopD. A: HiLoad 16/60 Superdex 200 elution profile of AcrH and SDS-PAGE showing crosslinking of AcrH. Lane (1) M w marker and lane (2) crosslinked product of AcrH. The major product
    Figure Legend Snippet: Purification and crosslinking of complexes formed between AcrH and AopB or AopD. A: HiLoad 16/60 Superdex 200 elution profile of AcrH and SDS-PAGE showing crosslinking of AcrH. Lane (1) M w marker and lane (2) crosslinked product of AcrH. The major product

    Techniques Used: Purification, SDS Page, Marker

    Limited protease digestion of the AcrH-AopD complex. A: SDS-PAGE showing partial digestion of AcrH-AopD with chymotrypsin. Lane (1) M w marker; lane (2) monomeric AcrH-AopD from gel filtration before digestion; and lane (3) AopD is being digested into
    Figure Legend Snippet: Limited protease digestion of the AcrH-AopD complex. A: SDS-PAGE showing partial digestion of AcrH-AopD with chymotrypsin. Lane (1) M w marker; lane (2) monomeric AcrH-AopD from gel filtration before digestion; and lane (3) AopD is being digested into

    Techniques Used: SDS Page, Marker, Filtration

    Purification of the AcrH-AopB-AopD and the AcrH-AopB 1–264 -AopD complexes. A: Purification of the AcrH-AopB-AopD complex. Lane (1) M w marker and lane (2) purified AcrH-AopB-AopD complex from Ni-NTA column with His-tag placed on AopD. The gel filtration
    Figure Legend Snippet: Purification of the AcrH-AopB-AopD and the AcrH-AopB 1–264 -AopD complexes. A: Purification of the AcrH-AopB-AopD complex. Lane (1) M w marker and lane (2) purified AcrH-AopB-AopD complex from Ni-NTA column with His-tag placed on AopD. The gel filtration

    Techniques Used: Purification, Marker, Filtration

    11) Product Images from "AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny"

    Article Title: AcMNPV Core Gene ac109 Is Required for Budded Virion Transport to the Nucleus and for Occlusion of Viral Progeny

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046146

    Detection of the entrance of ac109 knockout-baculovirus in insect cells. Immunofluorescence of insect cells infected with wild type (wt) Ac MNPV or with ac109 KO BVs. Sf-9 cells were incubated with wt Ac MNPV at a multiplicity of infection of 0.5 or supernatants of Bac- ac109 KO transfected cells for 45 min at 4°C. Then, cells were attached on culture dishes and incubated for 2 h at 27°C. Fixed cells were treated with an anti-VP39 monoclonal antibody and an Alexa Fluor® 488 conjugated anti-mouse antibody. Next, cells were stained with Alexa Fluor® 633 phalloidin and observed by confocal microscopy. Bars represent 10 µm.
    Figure Legend Snippet: Detection of the entrance of ac109 knockout-baculovirus in insect cells. Immunofluorescence of insect cells infected with wild type (wt) Ac MNPV or with ac109 KO BVs. Sf-9 cells were incubated with wt Ac MNPV at a multiplicity of infection of 0.5 or supernatants of Bac- ac109 KO transfected cells for 45 min at 4°C. Then, cells were attached on culture dishes and incubated for 2 h at 27°C. Fixed cells were treated with an anti-VP39 monoclonal antibody and an Alexa Fluor® 488 conjugated anti-mouse antibody. Next, cells were stained with Alexa Fluor® 633 phalloidin and observed by confocal microscopy. Bars represent 10 µm.

    Techniques Used: Knock-Out, Immunofluorescence, Infection, Incubation, BAC Assay, Transfection, Staining, Confocal Microscopy

    Ac109 localization in virions and in Sf-9 cells. (A) Western blot analysis of purified virions. Budded viruses (BVs), obtained from supernatants of Sf-9 cells infected with wild type (wt) Ac MNPV at a multiplicity of infection (MOI) of 0.1, were purified through a 25% w/v sucrose followed by a 25–65% sucrose gradient. Purified BVs were resuspended in PBS, and nucleocapsids and envelopes were separated using 1% IGEPAL and ultracentrifugation through a glycerol cushion. Occlusion derived viruses (ODVs) were obtained after alkaline treatment of polyhedra recovered from the infected Sf-9 cells. ODVs were purified through a discontinuous 30–65% sucrose gradient. Ac109, GP64 and VP39 proteins were detected from purified ODVs, BVs, BVs envelopes (Env) and nucleocapsid (Nc) fractions with a polyclonal serum anti Ac109-GST fusion protein, an anti-GP64 monoclonal antibody or an anti-VP39 monoclonal antibody. Sf-9 cells infected with wt Ac MNPV (IC) were used as a control of Ac109 detection. (B) Subcellular localization of Ac109 in living cells. Ac109 fused to YFP protein at either C or N-terminus (Ac109:YFP and YFP:Ac109) and transcribed from pOpIE2 promoter was detected in Sf-9.RNuc cells by confocal microscopy at 48 h post-transfection (hpt). (C) Kinetics of subcellular localization of Ac109 during baculovirus infection. Confocal microscopy of Sf-9.RNuc cells infected with Acppolp109-109Y virus at a MOI of 5 is shown at indicated h post-infection (hpi). Ac109 was fused to YFP at the C-terminal end (Ac109:YFP) and transcribed from the ac109 promoter. (D) Living insect cells infected with Acppolp109-109Y virus showing a heterogeneous Ac109:YFP accumulation around forming polyhedra in the nucleus. (E) Ac109:YFP fluorescence concentrated in the already formed polyhedra. (F) Cytoplasmic accumulations of Ac109:YFP in Sf-9.RNuc cells at different stages of viral infection. Bars represent 10 µm.
    Figure Legend Snippet: Ac109 localization in virions and in Sf-9 cells. (A) Western blot analysis of purified virions. Budded viruses (BVs), obtained from supernatants of Sf-9 cells infected with wild type (wt) Ac MNPV at a multiplicity of infection (MOI) of 0.1, were purified through a 25% w/v sucrose followed by a 25–65% sucrose gradient. Purified BVs were resuspended in PBS, and nucleocapsids and envelopes were separated using 1% IGEPAL and ultracentrifugation through a glycerol cushion. Occlusion derived viruses (ODVs) were obtained after alkaline treatment of polyhedra recovered from the infected Sf-9 cells. ODVs were purified through a discontinuous 30–65% sucrose gradient. Ac109, GP64 and VP39 proteins were detected from purified ODVs, BVs, BVs envelopes (Env) and nucleocapsid (Nc) fractions with a polyclonal serum anti Ac109-GST fusion protein, an anti-GP64 monoclonal antibody or an anti-VP39 monoclonal antibody. Sf-9 cells infected with wt Ac MNPV (IC) were used as a control of Ac109 detection. (B) Subcellular localization of Ac109 in living cells. Ac109 fused to YFP protein at either C or N-terminus (Ac109:YFP and YFP:Ac109) and transcribed from pOpIE2 promoter was detected in Sf-9.RNuc cells by confocal microscopy at 48 h post-transfection (hpt). (C) Kinetics of subcellular localization of Ac109 during baculovirus infection. Confocal microscopy of Sf-9.RNuc cells infected with Acppolp109-109Y virus at a MOI of 5 is shown at indicated h post-infection (hpi). Ac109 was fused to YFP at the C-terminal end (Ac109:YFP) and transcribed from the ac109 promoter. (D) Living insect cells infected with Acppolp109-109Y virus showing a heterogeneous Ac109:YFP accumulation around forming polyhedra in the nucleus. (E) Ac109:YFP fluorescence concentrated in the already formed polyhedra. (F) Cytoplasmic accumulations of Ac109:YFP in Sf-9.RNuc cells at different stages of viral infection. Bars represent 10 µm.

    Techniques Used: Western Blot, Purification, Infection, Derivative Assay, Confocal Microscopy, Transfection, Fluorescence

    12) Product Images from "Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila"

    Article Title: Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.187

    Purification and crosslinking of complexes formed between AcrH and AopB or AopD. A: HiLoad 16/60 Superdex 200 elution profile of AcrH and SDS-PAGE showing crosslinking of AcrH. Lane (1) M w marker and lane (2) crosslinked product of AcrH. The major product
    Figure Legend Snippet: Purification and crosslinking of complexes formed between AcrH and AopB or AopD. A: HiLoad 16/60 Superdex 200 elution profile of AcrH and SDS-PAGE showing crosslinking of AcrH. Lane (1) M w marker and lane (2) crosslinked product of AcrH. The major product

    Techniques Used: Purification, SDS Page, Marker

    Limited protease digestion of the AcrH-AopB complex. A: SDS-PAGE showing partial digestion of AcrH-AopB with elastase. Lane (1) M w marker; lane (2) monomeric AcrH-AopB from gel filtration before digestion; and lane (3) AopB is being digested into a single
    Figure Legend Snippet: Limited protease digestion of the AcrH-AopB complex. A: SDS-PAGE showing partial digestion of AcrH-AopB with elastase. Lane (1) M w marker; lane (2) monomeric AcrH-AopB from gel filtration before digestion; and lane (3) AopB is being digested into a single

    Techniques Used: SDS Page, Marker, Filtration

    Thermal denaturation of AcrH and various complexes as monitored by FarUV-CD at 222 nm. The fraction of protein denatured is plotted against temperature (°C). Legends for different proteins are: dimeric AcrH (open circle); monomeric AcrH-AopB (open
    Figure Legend Snippet: Thermal denaturation of AcrH and various complexes as monitored by FarUV-CD at 222 nm. The fraction of protein denatured is plotted against temperature (°C). Legends for different proteins are: dimeric AcrH (open circle); monomeric AcrH-AopB (open

    Techniques Used:

    Purification of the AcrH-AopB-AopD and the AcrH-AopB 1–264 -AopD complexes. A: Purification of the AcrH-AopB-AopD complex. Lane (1) M w marker and lane (2) purified AcrH-AopB-AopD complex from Ni-NTA column with His-tag placed on AopD. The gel filtration
    Figure Legend Snippet: Purification of the AcrH-AopB-AopD and the AcrH-AopB 1–264 -AopD complexes. A: Purification of the AcrH-AopB-AopD complex. Lane (1) M w marker and lane (2) purified AcrH-AopB-AopD complex from Ni-NTA column with His-tag placed on AopD. The gel filtration

    Techniques Used: Purification, Marker, Filtration

    13) Product Images from "Sublingual Immunization with M2-Based Vaccine Induces Broad Protective Immunity against Influenza"

    Article Title: Sublingual Immunization with M2-Based Vaccine Induces Broad Protective Immunity against Influenza

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027953

    Construction of plasmids and purification of M2 proteins. (A) The synthetic M2eC or 3M2eC genes without hydrophobic region (amino acids 26–55) from PR8 virus were cloned into pET15b vector (B). The recombinant proteins expressed in E. coli were purified by His-tag affinity chromatography and detected by Western blot using M2e-specific monoclonal Ab, 14C2.
    Figure Legend Snippet: Construction of plasmids and purification of M2 proteins. (A) The synthetic M2eC or 3M2eC genes without hydrophobic region (amino acids 26–55) from PR8 virus were cloned into pET15b vector (B). The recombinant proteins expressed in E. coli were purified by His-tag affinity chromatography and detected by Western blot using M2e-specific monoclonal Ab, 14C2.

    Techniques Used: Purification, Clone Assay, Plasmid Preparation, Recombinant, Affinity Chromatography, Western Blot

    14) Product Images from "Transcriptome-based analysis of putative allergens of Chorioptes texanus"

    Article Title: Transcriptome-based analysis of putative allergens of Chorioptes texanus

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-019-3843-7

    Hematoxylin and eosin (H  E) staining.  a  H  E staining of skin injected with recombinant Der p 1-like protein.  b  H  E staining of skin injected with recombinant Der p 7-like protein.  c  H  E staining of skin injected with recombinant Eur m 1-like protein. Arrows indicate eosinophils
    Figure Legend Snippet: Hematoxylin and eosin (H E) staining. a H E staining of skin injected with recombinant Der p 1-like protein. b H E staining of skin injected with recombinant Der p 7-like protein. c H E staining of skin injected with recombinant Eur m 1-like protein. Arrows indicate eosinophils

    Techniques Used: Staining, Injection, Recombinant

    Intradermal skin test.  a  Histamine (4 mg/ml, 0.1 ml).  b  400 μg of purified pET-32a (+) empty expression vector in 0.1 ml PBS.  c  0.1 ml PBS.  d  100 μg of purified recombinant Der p 1-like protein.  e  200 μg of purified recombinant Der p 1-like protein.  f  400 μg of purified recombinant Der p 1-like protein.  g  100 μg of purified recombinant Der p 7-like protein.  h  200 μg of purified recombinant Der p 7-like protein.  i  400 μg of purified recombinant Der p 7-like protein.  j  100 μg of purified recombinant Eur m 1-like protein.  k  200 μg of purified recombinant Eur m 1-like protein.  l  400 μg of purified recombinant Eur m 1-like protein
    Figure Legend Snippet: Intradermal skin test. a Histamine (4 mg/ml, 0.1 ml). b 400 μg of purified pET-32a (+) empty expression vector in 0.1 ml PBS. c 0.1 ml PBS. d 100 μg of purified recombinant Der p 1-like protein. e 200 μg of purified recombinant Der p 1-like protein. f 400 μg of purified recombinant Der p 1-like protein. g 100 μg of purified recombinant Der p 7-like protein. h 200 μg of purified recombinant Der p 7-like protein. i 400 μg of purified recombinant Der p 7-like protein. j 100 μg of purified recombinant Eur m 1-like protein. k 200 μg of purified recombinant Eur m 1-like protein. l 400 μg of purified recombinant Eur m 1-like protein

    Techniques Used: Purification, Positron Emission Tomography, Expressing, Plasmid Preparation, Recombinant

    15) Product Images from "Transcriptome-based analysis of putative allergens of Chorioptes texanus"

    Article Title: Transcriptome-based analysis of putative allergens of Chorioptes texanus

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-019-3843-7

    Hematoxylin and eosin (H  E) staining.  a  H  E staining of skin injected with recombinant Der p 1-like protein.  b  H  E staining of skin injected with recombinant Der p 7-like protein.  c  H  E staining of skin injected with recombinant Eur m 1-like protein. Arrows indicate eosinophils
    Figure Legend Snippet: Hematoxylin and eosin (H E) staining. a H E staining of skin injected with recombinant Der p 1-like protein. b H E staining of skin injected with recombinant Der p 7-like protein. c H E staining of skin injected with recombinant Eur m 1-like protein. Arrows indicate eosinophils

    Techniques Used: Staining, Injection, Recombinant

    Intradermal skin test.  a  Histamine (4 mg/ml, 0.1 ml).  b  400 μg of purified pET-32a (+) empty expression vector in 0.1 ml PBS.  c  0.1 ml PBS.  d  100 μg of purified recombinant Der p 1-like protein.  e  200 μg of purified recombinant Der p 1-like protein.  f  400 μg of purified recombinant Der p 1-like protein.  g  100 μg of purified recombinant Der p 7-like protein.  h  200 μg of purified recombinant Der p 7-like protein.  i  400 μg of purified recombinant Der p 7-like protein.  j  100 μg of purified recombinant Eur m 1-like protein.  k  200 μg of purified recombinant Eur m 1-like protein.  l  400 μg of purified recombinant Eur m 1-like protein
    Figure Legend Snippet: Intradermal skin test. a Histamine (4 mg/ml, 0.1 ml). b 400 μg of purified pET-32a (+) empty expression vector in 0.1 ml PBS. c 0.1 ml PBS. d 100 μg of purified recombinant Der p 1-like protein. e 200 μg of purified recombinant Der p 1-like protein. f 400 μg of purified recombinant Der p 1-like protein. g 100 μg of purified recombinant Der p 7-like protein. h 200 μg of purified recombinant Der p 7-like protein. i 400 μg of purified recombinant Der p 7-like protein. j 100 μg of purified recombinant Eur m 1-like protein. k 200 μg of purified recombinant Eur m 1-like protein. l 400 μg of purified recombinant Eur m 1-like protein

    Techniques Used: Purification, Positron Emission Tomography, Expressing, Plasmid Preparation, Recombinant

    16) Product Images from "Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis"

    Article Title: Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013143

    Purification of the Rv0045c protein by ion exchange chromatography and gel filtration chromatography. The Rv0045c protein was purified by anion exchange chromatography (A), cation exchange chromatography (B), and gel filtration chromatography (C). The purity was checked by SDS-PAGE analysis after each purification procedure.
    Figure Legend Snippet: Purification of the Rv0045c protein by ion exchange chromatography and gel filtration chromatography. The Rv0045c protein was purified by anion exchange chromatography (A), cation exchange chromatography (B), and gel filtration chromatography (C). The purity was checked by SDS-PAGE analysis after each purification procedure.

    Techniques Used: Purification, Ion Exchange Chromatography, Filtration, Chromatography, SDS Page

    MALDI-TOF peptide mass fingerprint (PMF) spectrometry of the Rv0045c protein. The PMF analysis was made from fragments of purified Rv0045c protein derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments was shown in bold red.
    Figure Legend Snippet: MALDI-TOF peptide mass fingerprint (PMF) spectrometry of the Rv0045c protein. The PMF analysis was made from fragments of purified Rv0045c protein derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments was shown in bold red.

    Techniques Used: Peptide Mass Fingerprinting, Purification, Derivative Assay, Sequencing

    CD spectra of the Rv0045c protein at different pH and temperatures. The CD measurements were made in the presence of various pH (A) at pH 2.0 (black), pH 3.0 (red), pH 4.0 (yellow), pH 6.0 (blue), pH 7.0 (purple), pH 8.0 (pink), pH 9.0 (green), pH 10.0 (gray), pH 11.0 (coral) and pH 12.0 (light green) at room temperature, and different temperatures (B) at 10°C (black), 20°C (gray), 30°C (yellow), 40°C (green), 50°C (blue), 60°C (purple) and 70°C (red) at pH 7.5, respectively. Values represent the mean ± SD of three analyses. The concentration of the Rv0045c protein was fixed at 0.35 mg/mL (20 mM Tris, pH 7.5).
    Figure Legend Snippet: CD spectra of the Rv0045c protein at different pH and temperatures. The CD measurements were made in the presence of various pH (A) at pH 2.0 (black), pH 3.0 (red), pH 4.0 (yellow), pH 6.0 (blue), pH 7.0 (purple), pH 8.0 (pink), pH 9.0 (green), pH 10.0 (gray), pH 11.0 (coral) and pH 12.0 (light green) at room temperature, and different temperatures (B) at 10°C (black), 20°C (gray), 30°C (yellow), 40°C (green), 50°C (blue), 60°C (purple) and 70°C (red) at pH 7.5, respectively. Values represent the mean ± SD of three analyses. The concentration of the Rv0045c protein was fixed at 0.35 mg/mL (20 mM Tris, pH 7.5).

    Techniques Used: Concentration Assay

    Effects of temperature and pH on enzyme activity of the Rv0045c protein. The enzyme activities were measured using p-butyrate caprylate (C 6 ) as substrate in the presence of mild temperatures (36°C–40°C) at pH 6.0 (red), pH 7.0 (green) and pH 8.0 (blue). Values represent the mean ± SD of five analyses. The concentration of the Rvoo45c protein was fixed at 0.2 mg/mL (20 mM Tris, pH 7.5). The enzyme activities were expressed as units hydrolase/mg protein/min (one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min).
    Figure Legend Snippet: Effects of temperature and pH on enzyme activity of the Rv0045c protein. The enzyme activities were measured using p-butyrate caprylate (C 6 ) as substrate in the presence of mild temperatures (36°C–40°C) at pH 6.0 (red), pH 7.0 (green) and pH 8.0 (blue). Values represent the mean ± SD of five analyses. The concentration of the Rvoo45c protein was fixed at 0.2 mg/mL (20 mM Tris, pH 7.5). The enzyme activities were expressed as units hydrolase/mg protein/min (one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min).

    Techniques Used: Activity Assay, Concentration Assay

    SDS-PAGE analysis for expression and affinity chromatography of the Rv0045c protein. Lane 1, culture pellet (uninduced); Lane 2, culture pellet (induced with 0.3 mM IPTG at 16°C); Lane 3, the supernatant of induced cells after sonication; Lane 4, fluid through Ni 2+ -affinity chromatography column; Lane 5 and 7: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 200 mM Imidazole, pH 7.5; Lane 6 and 8: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 500 mM Imidazole, pH 7.5; Lane M: molecular mass markers.
    Figure Legend Snippet: SDS-PAGE analysis for expression and affinity chromatography of the Rv0045c protein. Lane 1, culture pellet (uninduced); Lane 2, culture pellet (induced with 0.3 mM IPTG at 16°C); Lane 3, the supernatant of induced cells after sonication; Lane 4, fluid through Ni 2+ -affinity chromatography column; Lane 5 and 7: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 200 mM Imidazole, pH 7.5; Lane 6 and 8: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 500 mM Imidazole, pH 7.5; Lane M: molecular mass markers.

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

    17) Product Images from "An ancient satellite repeat controls gene expression and embryonic development in Aedes aegypti through a highly conserved piRNA"

    Article Title: An ancient satellite repeat controls gene expression and embryonic development in Aedes aegypti through a highly conserved piRNA

    Journal: bioRxiv

    doi: 10.1101/2020.01.15.907428

    An antisense oligonucleotide relieves tapiR1-mediated silencing. (A) Luciferase assay of a reporter with a fully complementary target site for tapiR1 in the 3’ UTR. Cells were co-transfected with the reporter and increasing amounts of a fully 2’ O -methylated antisense RNA oligonucleotide (AO), or a control AO. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter. Indicated are mean, standard deviation and individual measurements from a representative experiment measured in triplicate. (B) Northern blot detection of tapiR1 in Aag2 cells upon treatment with tapiR1 or control AO in Aag2 cells. Cells were harvested after the indicated time points. Ethidium bromide-stained rRNA serves as loading control.
    Figure Legend Snippet: An antisense oligonucleotide relieves tapiR1-mediated silencing. (A) Luciferase assay of a reporter with a fully complementary target site for tapiR1 in the 3’ UTR. Cells were co-transfected with the reporter and increasing amounts of a fully 2’ O -methylated antisense RNA oligonucleotide (AO), or a control AO. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter. Indicated are mean, standard deviation and individual measurements from a representative experiment measured in triplicate. (B) Northern blot detection of tapiR1 in Aag2 cells upon treatment with tapiR1 or control AO in Aag2 cells. Cells were harvested after the indicated time points. Ethidium bromide-stained rRNA serves as loading control.

    Techniques Used: Luciferase, Transfection, Methylation, Activity Assay, Standard Deviation, Northern Blot, Staining

    tapiR silences gene expression in Aag2 cells. (A) log2 mRNA expression of transposable elements in Aag2 cells treated with a tapiR1 specific antisense oligonucleotide (AO) or control AO. Depicted are the means of three biological replicates. A pseudo-count of one was added to all values in order to plot values of zero. Diagonal lines represent a fold change of two. Significance was tested at an FDR of 0.01 and a log2 fold change of 0.5. (B) log2 fold changes of genes upon treatment with tapiR1 or control AO in Aag2 cells (left) and mosquito embryos (right) plotted against the minimum free energy of predicted tapiR1-target duplexes. Blue dots indicate target sites that were confirmed to be functional, and red dots indicate target sites that were not functional in luciferase reporter assays (see Extended Data Fig 8 ). (C) Violin plot of log2 fold changes of all genes in Aag2 cells (left) and mosquito embryos (right), either with or without predicted tapiR1 target site.
    Figure Legend Snippet: tapiR silences gene expression in Aag2 cells. (A) log2 mRNA expression of transposable elements in Aag2 cells treated with a tapiR1 specific antisense oligonucleotide (AO) or control AO. Depicted are the means of three biological replicates. A pseudo-count of one was added to all values in order to plot values of zero. Diagonal lines represent a fold change of two. Significance was tested at an FDR of 0.01 and a log2 fold change of 0.5. (B) log2 fold changes of genes upon treatment with tapiR1 or control AO in Aag2 cells (left) and mosquito embryos (right) plotted against the minimum free energy of predicted tapiR1-target duplexes. Blue dots indicate target sites that were confirmed to be functional, and red dots indicate target sites that were not functional in luciferase reporter assays (see Extended Data Fig 8 ). (C) Violin plot of log2 fold changes of all genes in Aag2 cells (left) and mosquito embryos (right), either with or without predicted tapiR1 target site.

    Techniques Used: Expressing, Functional Assay, Luciferase

    Antibody validation, uncropped Western blot images, knockdown efficiencies, and scoring scheme for the development of Ae. aegypti embryos. (A) Validation of Ae. aegypti PIWI antibodies. Specificity was confirmed by detection of an additional band in PTH-tagged PIWI-expressing Aag2 cells, and loss of signal upon dsRNA-mediated knockdown. Knockdown with dsRNA targeting RLuc (dsRLuc) serves as negative control. (B) Uncropped Western blot images corresponding to Extended Data Fig 2B . (C-E) Knockdown efficiencies of PIWI genes shown in Extended Data Fig 2D (D) , siRNA and miRNA pathway genes shown in Extended Data Fig 2E (E) , and AAEL017385 isoforms in the experiment shown in Extended Data Fig 3C (F) (F) Representative images of embryos scored as either undeveloped, intermediate or fully developed at 2.5 days post injection with antisense RNA oligonucleotides.
    Figure Legend Snippet: Antibody validation, uncropped Western blot images, knockdown efficiencies, and scoring scheme for the development of Ae. aegypti embryos. (A) Validation of Ae. aegypti PIWI antibodies. Specificity was confirmed by detection of an additional band in PTH-tagged PIWI-expressing Aag2 cells, and loss of signal upon dsRNA-mediated knockdown. Knockdown with dsRNA targeting RLuc (dsRLuc) serves as negative control. (B) Uncropped Western blot images corresponding to Extended Data Fig 2B . (C-E) Knockdown efficiencies of PIWI genes shown in Extended Data Fig 2D (D) , siRNA and miRNA pathway genes shown in Extended Data Fig 2E (E) , and AAEL017385 isoforms in the experiment shown in Extended Data Fig 3C (F) (F) Representative images of embryos scored as either undeveloped, intermediate or fully developed at 2.5 days post injection with antisense RNA oligonucleotides.

    Techniques Used: Western Blot, Expressing, Negative Control, Injection

    tapiR1 silences target RNAs in trans through seed-mediated base pairing. (A) Schematic representation of the firefly luciferase (FLuc) reporter constructs (left panel) and luciferase assay in Aag2 cells transfected with reporters containing no target site (empty), a fully complementary target site to tapiR1, or a control target site. (B) Luciferase assay of reporters with tapiR1 target sites, mismatched sites (mm4), or control sequences located at different positions in the reporter mRNA. (C, D) Luciferase assay of tapiR1 reporters harbouring three consecutive mismatches (C), or increasing number of mismatches (D) at the indicated positions of the piRNA target site in the 3’ UTR of firefly luciferase. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter. Indicated are mean, standard deviation, and individual measurements of a representative experiment performed with two to three independent clones per construct and measured in triplicates. (E) log2 expression of mRNAs and lncRNAs in Aag2 cells upon treatment with a tapiR1 specific or control antisense oligonucleotide (AO). Depicted are average read counts in three biological replicates. A pseudo-count of one was added to all values in order to plot values of zero. Diagonal lines indicate a fold change of two. Significance was tested at a false discovery rate (FDR) of 0.01 and a log2 fold change of 0.5 as indicated by coloured dots. (F) RT-qPCR of tapiR1 target genes upon transfection of Aag2 cells with tapiR1 specific or control AO. Depicted are mean, standard deviation, and individual measurements of one experiment measured in technical duplicates.
    Figure Legend Snippet: tapiR1 silences target RNAs in trans through seed-mediated base pairing. (A) Schematic representation of the firefly luciferase (FLuc) reporter constructs (left panel) and luciferase assay in Aag2 cells transfected with reporters containing no target site (empty), a fully complementary target site to tapiR1, or a control target site. (B) Luciferase assay of reporters with tapiR1 target sites, mismatched sites (mm4), or control sequences located at different positions in the reporter mRNA. (C, D) Luciferase assay of tapiR1 reporters harbouring three consecutive mismatches (C), or increasing number of mismatches (D) at the indicated positions of the piRNA target site in the 3’ UTR of firefly luciferase. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter. Indicated are mean, standard deviation, and individual measurements of a representative experiment performed with two to three independent clones per construct and measured in triplicates. (E) log2 expression of mRNAs and lncRNAs in Aag2 cells upon treatment with a tapiR1 specific or control antisense oligonucleotide (AO). Depicted are average read counts in three biological replicates. A pseudo-count of one was added to all values in order to plot values of zero. Diagonal lines indicate a fold change of two. Significance was tested at a false discovery rate (FDR) of 0.01 and a log2 fold change of 0.5 as indicated by coloured dots. (F) RT-qPCR of tapiR1 target genes upon transfection of Aag2 cells with tapiR1 specific or control AO. Depicted are mean, standard deviation, and individual measurements of one experiment measured in technical duplicates.

    Techniques Used: Luciferase, Construct, Transfection, Activity Assay, Standard Deviation, Clone Assay, Expressing, Quantitative RT-PCR

    Related Articles

    Clone Assay:

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    Article Snippet: .. VHH Antibody Expression and Purification After double digestion with BamHI and HindIII enzymes, the VHH genes from the selected clones were ligated into pET-22b (+) vector and transformed into E. coli BL21 (DE3) (Novagen, Madison, WI). .. Large scale production of recombinant VHHs was performed by culturing the bacteria in LB medium supplemented with ampicillin (1∶1000) until the OD600 reached 0.6–0.8.

    Article Title: MazF6 toxin of Mycobacterium tuberculosis demonstrates antitoxin specificity and is coupled to regulation of cell growth by a Soj-like protein
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    Centrifugation:

    Article Title: Mutational specificity and genetic control of replicative bypass of an abasic site in yeast
    Article Snippet: .. Monoadducted heteroduplex plasmids were generated as follows: Plasmids pVP9 and pVP10 were digested with BamHI and PstI ( A ), and the vector portion was separated from the 25-bp insert by centrifugation in a YM-30 microcon filter unit (Millipore). ..

    Positron Emission Tomography:

    Article Title: Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis
    Article Snippet: .. VHH Antibody Expression and Purification After double digestion with BamHI and HindIII enzymes, the VHH genes from the selected clones were ligated into pET-22b (+) vector and transformed into E. coli BL21 (DE3) (Novagen, Madison, WI). .. Large scale production of recombinant VHHs was performed by culturing the bacteria in LB medium supplemented with ampicillin (1∶1000) until the OD600 reached 0.6–0.8.

    Agarose Gel Electrophoresis:

    Article Title: Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response
    Article Snippet: .. Five micrograms of resultant DNA were digested with BamHI (which does not cut the HPV31 genome) or HindIII (which linearizes the HPV31 genome), separated on 0.8% agarose gel for 15 h at 40 V, and subsequently transferred to a positively charged nylon membrane (Immobilon-Ny+; Millipore). ..

    Synthesized:

    Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
    Article Snippet: .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

    Transfection:

    Article Title: Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host
    Article Snippet: .. This construct was subcloned into the BamH I/Xho I sites of the pET21a plasmid (Novagen) and transfected into E. coli BL21 (DE3). .. In order to express and obtain rSm16, transformed cells were cultured overnight at 37°C in liquid LB medium (Kasvi) supplemented with 100 μ g/ml ampicillin (Sigma-Aldrich).

    Construct:

    Article Title: Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host
    Article Snippet: .. This construct was subcloned into the BamH I/Xho I sites of the pET21a plasmid (Novagen) and transfected into E. coli BL21 (DE3). .. In order to express and obtain rSm16, transformed cells were cultured overnight at 37°C in liquid LB medium (Kasvi) supplemented with 100 μ g/ml ampicillin (Sigma-Aldrich).

    Purification:

    Article Title: Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis
    Article Snippet: .. VHH Antibody Expression and Purification After double digestion with BamHI and HindIII enzymes, the VHH genes from the selected clones were ligated into pET-22b (+) vector and transformed into E. coli BL21 (DE3) (Novagen, Madison, WI). .. Large scale production of recombinant VHHs was performed by culturing the bacteria in LB medium supplemented with ampicillin (1∶1000) until the OD600 reached 0.6–0.8.

    Generated:

    Article Title: Mutational specificity and genetic control of replicative bypass of an abasic site in yeast
    Article Snippet: .. Monoadducted heteroduplex plasmids were generated as follows: Plasmids pVP9 and pVP10 were digested with BamHI and PstI ( A ), and the vector portion was separated from the 25-bp insert by centrifugation in a YM-30 microcon filter unit (Millipore). ..

    other:

    Article Title: Single-Molecule Approach to Bacterial Genomic Comparisons via Optical Mapping †
    Article Snippet: The surface properties were assayed by digesting lambda DASH II bacteriophage DNA with 40 U of BamHI diluted in 100 μl of digestion buffer with 0.02% Triton X-100 (Sigma) at 37°C to determine optimal digestion times, which ranged from 40 to 120 min.

    Expressing:

    Article Title: Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis
    Article Snippet: .. VHH Antibody Expression and Purification After double digestion with BamHI and HindIII enzymes, the VHH genes from the selected clones were ligated into pET-22b (+) vector and transformed into E. coli BL21 (DE3) (Novagen, Madison, WI). .. Large scale production of recombinant VHHs was performed by culturing the bacteria in LB medium supplemented with ampicillin (1∶1000) until the OD600 reached 0.6–0.8.

    Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
    Article Snippet: .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

    Sequencing:

    Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
    Article Snippet: .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

    Transformation Assay:

    Article Title: Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis
    Article Snippet: .. VHH Antibody Expression and Purification After double digestion with BamHI and HindIII enzymes, the VHH genes from the selected clones were ligated into pET-22b (+) vector and transformed into E. coli BL21 (DE3) (Novagen, Madison, WI). .. Large scale production of recombinant VHHs was performed by culturing the bacteria in LB medium supplemented with ampicillin (1∶1000) until the OD600 reached 0.6–0.8.

    Plasmid Preparation:

    Article Title: Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis
    Article Snippet: .. VHH Antibody Expression and Purification After double digestion with BamHI and HindIII enzymes, the VHH genes from the selected clones were ligated into pET-22b (+) vector and transformed into E. coli BL21 (DE3) (Novagen, Madison, WI). .. Large scale production of recombinant VHHs was performed by culturing the bacteria in LB medium supplemented with ampicillin (1∶1000) until the OD600 reached 0.6–0.8.

    Article Title: Mutational specificity and genetic control of replicative bypass of an abasic site in yeast
    Article Snippet: .. Monoadducted heteroduplex plasmids were generated as follows: Plasmids pVP9 and pVP10 were digested with BamHI and PstI ( A ), and the vector portion was separated from the 25-bp insert by centrifugation in a YM-30 microcon filter unit (Millipore). ..

    Article Title: Sm16, A Schistosoma mansoni Immunomodulatory Protein, Fails to Elicit a Protective Immune Response and Does Not Have an Essential Role in Parasite Survival in the Definitive Host
    Article Snippet: .. This construct was subcloned into the BamH I/Xho I sites of the pET21a plasmid (Novagen) and transfected into E. coli BL21 (DE3). .. In order to express and obtain rSm16, transformed cells were cultured overnight at 37°C in liquid LB medium (Kasvi) supplemented with 100 μ g/ml ampicillin (Sigma-Aldrich).

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    Millipore prp s43a
    Purification of phosphorylated PrP from PrP/Cdk5/p25 co-transfected N2a cells protein extracts A-D. Western blot analyses with 3F4, phospho-tyrosine (pTyr), anti-pPrP S43 and β-actin on phospho-column fractionated proteins from PrP/Cdk5/p25 co-transfected N2a cells ( A ), roscovitine-treated PrP/Cdk5/p25 co-transfected N2a cells ( B ), PrP <t>S43A/Cdk5/p25</t> co-transfected N2a cells ( C ), PrP-transfected N2a ( D ), or untransfected N2a cells ( E ).
    Prp S43a, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore pet28a
    Purification of phosphorylated PrP from PrP/Cdk5/p25 co-transfected N2a cells protein extracts A-D. Western blot analyses with 3F4, phospho-tyrosine (pTyr), anti-pPrP S43 and β-actin on phospho-column fractionated proteins from PrP/Cdk5/p25 co-transfected N2a cells ( A ), roscovitine-treated PrP/Cdk5/p25 co-transfected N2a cells ( B ), PrP <t>S43A/Cdk5/p25</t> co-transfected N2a cells ( C ), PrP-transfected N2a ( D ), or untransfected N2a cells ( E ).
    Pet28a, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Purification of phosphorylated PrP from PrP/Cdk5/p25 co-transfected N2a cells protein extracts A-D. Western blot analyses with 3F4, phospho-tyrosine (pTyr), anti-pPrP S43 and β-actin on phospho-column fractionated proteins from PrP/Cdk5/p25 co-transfected N2a cells ( A ), roscovitine-treated PrP/Cdk5/p25 co-transfected N2a cells ( B ), PrP S43A/Cdk5/p25 co-transfected N2a cells ( C ), PrP-transfected N2a ( D ), or untransfected N2a cells ( E ).

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Phosphorylation of Prion Protein at Serine 43 Induces Prion Protein Conformational Change

    doi: 10.1523/JNEUROSCI.2294-09.2009

    Figure Lengend Snippet: Purification of phosphorylated PrP from PrP/Cdk5/p25 co-transfected N2a cells protein extracts A-D. Western blot analyses with 3F4, phospho-tyrosine (pTyr), anti-pPrP S43 and β-actin on phospho-column fractionated proteins from PrP/Cdk5/p25 co-transfected N2a cells ( A ), roscovitine-treated PrP/Cdk5/p25 co-transfected N2a cells ( B ), PrP S43A/Cdk5/p25 co-transfected N2a cells ( C ), PrP-transfected N2a ( D ), or untransfected N2a cells ( E ).

    Article Snippet: PrP and PrP S43A were subcloned into the BamH I and Xho I sites of the pET-23b(+) vector (EMD Chemicals, Gibbstown, NJ) after PCR amplification with the forward primer 5′-ACGCGGATCCCAAGAAGCGCCCGAAGCCT-3′ and the reverse primer 5′-GCCGCTCGAGGCTCGATCCTCTCTGGTA-3′.

    Techniques: Purification, Transfection, Western Blot