Structured Review

Nacalai retentate
Effect of pH on homomultimer formation of His-tagged Orf8 and Orf16 proteins. The recombinant proteins were incubated at various pH values and cross-linked with glutaraldehyde. The samples (250 ng/lane) were electrophoresed and CBB stained (A). The recombinant proteins were incubated at pH 2.5 and pH 7.5. After ultrafiltration (100-kDa cutoff), permeate (P) and <t>retentate</t> (R) fractions were electrophoresed and CBB stained (B). To ascertain pH stability, the recombinant proteins were incubated at various pH values, diluted, and shifted to pH 7–8. After cross-linking, the samples (25 ng/lane) were electrophoresed and silver stained (C). In all panels, large closed and open triangles indicate multimers and monomers, respectively.
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Images

1) Product Images from "Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T"

Article Title: Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706T

Journal: Meta Gene

doi: 10.1016/j.mgene.2015.03.001

Effect of pH on homomultimer formation of His-tagged Orf8 and Orf16 proteins. The recombinant proteins were incubated at various pH values and cross-linked with glutaraldehyde. The samples (250 ng/lane) were electrophoresed and CBB stained (A). The recombinant proteins were incubated at pH 2.5 and pH 7.5. After ultrafiltration (100-kDa cutoff), permeate (P) and retentate (R) fractions were electrophoresed and CBB stained (B). To ascertain pH stability, the recombinant proteins were incubated at various pH values, diluted, and shifted to pH 7–8. After cross-linking, the samples (25 ng/lane) were electrophoresed and silver stained (C). In all panels, large closed and open triangles indicate multimers and monomers, respectively.
Figure Legend Snippet: Effect of pH on homomultimer formation of His-tagged Orf8 and Orf16 proteins. The recombinant proteins were incubated at various pH values and cross-linked with glutaraldehyde. The samples (250 ng/lane) were electrophoresed and CBB stained (A). The recombinant proteins were incubated at pH 2.5 and pH 7.5. After ultrafiltration (100-kDa cutoff), permeate (P) and retentate (R) fractions were electrophoresed and CBB stained (B). To ascertain pH stability, the recombinant proteins were incubated at various pH values, diluted, and shifted to pH 7–8. After cross-linking, the samples (25 ng/lane) were electrophoresed and silver stained (C). In all panels, large closed and open triangles indicate multimers and monomers, respectively.

Techniques Used: Recombinant, Incubation, Staining

2) Product Images from "Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana"

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana

Journal: Annals of Botany

doi: 10.1093/aob/mcy125

Hexahistidine-tagged CAM1 (His-CAM1) interacts with GST-fused group I bZIP proteins and their derivatives in vitro . His-CAM1 and GST-fused proteins were expressed in Escherichia coli , purified, and used for in vitro crosslink assays. For each panel, the
Figure Legend Snippet: Hexahistidine-tagged CAM1 (His-CAM1) interacts with GST-fused group I bZIP proteins and their derivatives in vitro . His-CAM1 and GST-fused proteins were expressed in Escherichia coli , purified, and used for in vitro crosslink assays. For each panel, the

Techniques Used: In Vitro, Purification

3) Product Images from "Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN"

Article Title: Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN

Journal: Acta Crystallographica. Section F, Structural Biology Communications

doi: 10.1107/S2053230X14028143

Structure of oxidized LGN in complex with Frmpd4. ( a ) Superposition of the structure of Frmpd4-L-bound LGN-N in an oxidized form with that of the LGN-N–Frmpd4-L complex. Frmpd4 and LGN in the oxidized complex are coloured blue and violet, respectively; Frmpd4 and LGN in the reduced complex are shown in green and grey, respectively. ( b ) Enlarged views of LGN TPR1 in the oxidized (upper) and reduced (lower) forms. Cys28 and Cys33 are shown as ball-and-stick models with electron density contoured at 1σ. ( c ) Interaction of LGN with Frmpd4 under reduced and oxidized conditions. LGN-N binding to MBP–Frmpd4-L in the presence of dithiothreitol (DTT) or H 2 O 2 was estimated by the MBP pull-down assay.
Figure Legend Snippet: Structure of oxidized LGN in complex with Frmpd4. ( a ) Superposition of the structure of Frmpd4-L-bound LGN-N in an oxidized form with that of the LGN-N–Frmpd4-L complex. Frmpd4 and LGN in the oxidized complex are coloured blue and violet, respectively; Frmpd4 and LGN in the reduced complex are shown in green and grey, respectively. ( b ) Enlarged views of LGN TPR1 in the oxidized (upper) and reduced (lower) forms. Cys28 and Cys33 are shown as ball-and-stick models with electron density contoured at 1σ. ( c ) Interaction of LGN with Frmpd4 under reduced and oxidized conditions. LGN-N binding to MBP–Frmpd4-L in the presence of dithiothreitol (DTT) or H 2 O 2 was estimated by the MBP pull-down assay.

Techniques Used: Binding Assay, Pull Down Assay

4) Product Images from "Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity"

Article Title: Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity

Journal: Journal of Virology

doi: 10.1128/JVI.01784-17

Translocation of VP6 mutants in the presence of VP3. (A) Colocalization of VP6 to which EGFP was fused at the C terminus with NS2 in the presence of VP3. Each of the mammalian expression plasmids, pCAG-PBTV10-VP6/N 87 C 115 (left) and pCAG-PBTV10-VP6/N 87 (right), was cotransfected with either pCAG-PBTV1-NS2 alone (middle) or pCAG-PBTV1-NS2 with pCAG-PBTV1-VP3 (bottom) into WT-BSR cells. As a control, pCAG-PBTV10-VP6/N 87 C 115 and pCAG-PBTV10-VP6/N 87 was each transfected alone into the cells (top). At 24 h posttransfection, the distributions of VP6-EGFP were observed using fluorescence microscopy. NS2 was detected using a guinea pig anti-NS2 antibody. Arrowheads, colocalization of VP6 with NS2. Nuclei were detected using DAPI. (B) Colocalization of VP6 to which EGFP was fused at the C terminus with VP3. Either VP6/N 87 C 115 (left) or VP6/N 87 (right) was coexpressed with VP3, using mammalian expression plasmids, in the absence (top) or presence of NS2 (bottom) in WT-BSR cells. The distributions of VP6-EGFP were observed using fluorescence microscopy. VP3 was detected using a mouse anti-VP3 antibody. Nuclei were detected using DAPI. Arrowheads, colocalization of VP6-EGFP and VP3.
Figure Legend Snippet: Translocation of VP6 mutants in the presence of VP3. (A) Colocalization of VP6 to which EGFP was fused at the C terminus with NS2 in the presence of VP3. Each of the mammalian expression plasmids, pCAG-PBTV10-VP6/N 87 C 115 (left) and pCAG-PBTV10-VP6/N 87 (right), was cotransfected with either pCAG-PBTV1-NS2 alone (middle) or pCAG-PBTV1-NS2 with pCAG-PBTV1-VP3 (bottom) into WT-BSR cells. As a control, pCAG-PBTV10-VP6/N 87 C 115 and pCAG-PBTV10-VP6/N 87 was each transfected alone into the cells (top). At 24 h posttransfection, the distributions of VP6-EGFP were observed using fluorescence microscopy. NS2 was detected using a guinea pig anti-NS2 antibody. Arrowheads, colocalization of VP6 with NS2. Nuclei were detected using DAPI. (B) Colocalization of VP6 to which EGFP was fused at the C terminus with VP3. Either VP6/N 87 C 115 (left) or VP6/N 87 (right) was coexpressed with VP3, using mammalian expression plasmids, in the absence (top) or presence of NS2 (bottom) in WT-BSR cells. The distributions of VP6-EGFP were observed using fluorescence microscopy. VP3 was detected using a mouse anti-VP3 antibody. Nuclei were detected using DAPI. Arrowheads, colocalization of VP6-EGFP and VP3.

Techniques Used: Translocation Assay, Expressing, Transfection, Fluorescence, Microscopy

Colocalization of VP3 with NS2. VP3 was coexpressed with NS2 in WT-BSR cells (bottom). As a control, either VP3 (top) or NS2 (middle) was singly expressed in the cells. VP3 and NS2 were detected using a mouse anti-VP3 antibody and a guinea pig anti-NS2 antibody, respectively.
Figure Legend Snippet: Colocalization of VP3 with NS2. VP3 was coexpressed with NS2 in WT-BSR cells (bottom). As a control, either VP3 (top) or NS2 (middle) was singly expressed in the cells. VP3 and NS2 were detected using a mouse anti-VP3 antibody and a guinea pig anti-NS2 antibody, respectively.

Techniques Used:

Localization of VP6, VP3, and NS2 in BTV-infected cells. WT-BSR cells were infected with each of WT-BTV (A), RA-BTV (B), RYF/3A-BTV (C), and d278/287-BTV (D), expressing WT VP6, RA VP6, RYF/3A VP6, and d278/287 VP6, respectively. Note that the cells were infected with WT-BTV and RA-BTV at an MOI of 0.1. At 24 h postinfection, the expression of VP6, VP3, and NS2 was observed using confocal microscopy. VP6 was detected using a guinea pig anti-VP6 antibody (top) and a rabbit anti-VP6 antibody (middle). VP3 was detected using a mouse anti-VP3 antibody. NS2 was detected using a guinea pig anti-NS2 antibody.
Figure Legend Snippet: Localization of VP6, VP3, and NS2 in BTV-infected cells. WT-BSR cells were infected with each of WT-BTV (A), RA-BTV (B), RYF/3A-BTV (C), and d278/287-BTV (D), expressing WT VP6, RA VP6, RYF/3A VP6, and d278/287 VP6, respectively. Note that the cells were infected with WT-BTV and RA-BTV at an MOI of 0.1. At 24 h postinfection, the expression of VP6, VP3, and NS2 was observed using confocal microscopy. VP6 was detected using a guinea pig anti-VP6 antibody (top) and a rabbit anti-VP6 antibody (middle). VP3 was detected using a mouse anti-VP3 antibody. NS2 was detected using a guinea pig anti-NS2 antibody.

Techniques Used: Infection, Expressing, Confocal Microscopy

Mapping of the amino acid residues of VP6 essential for VP3 binding. (A) Schematic representation of the changes introduced into Flag-VP6. The name of the mutation is indicated on the left. Numbers indicate amino acid positions according to the VP6 amino acid sequence. The amino acid changes are also shown. Asterisks indicate no change, and dashes indicate deletions. (B) The interaction between HA-VP3 and a series of Flag-VP6 mutants was analyzed by immunoprecipitation using either anti-HA MAb (top) or anti-Flag MAb (bottom). (C) The relative densities of Flag-VP6 mutant binding to HA-VP3 were quantified using gray-value analysis and normalized by the relative densities of HA-VP3. The activity of binding of each Flag-VP6 mutant to HA-VP3 was calculated in five experiments, and the results (means ± SDs) are shown as a percentage of the binding of the WT. *, a significant difference in comparison to the binding activity of WT Flag-VP6 ( P
Figure Legend Snippet: Mapping of the amino acid residues of VP6 essential for VP3 binding. (A) Schematic representation of the changes introduced into Flag-VP6. The name of the mutation is indicated on the left. Numbers indicate amino acid positions according to the VP6 amino acid sequence. The amino acid changes are also shown. Asterisks indicate no change, and dashes indicate deletions. (B) The interaction between HA-VP3 and a series of Flag-VP6 mutants was analyzed by immunoprecipitation using either anti-HA MAb (top) or anti-Flag MAb (bottom). (C) The relative densities of Flag-VP6 mutant binding to HA-VP3 were quantified using gray-value analysis and normalized by the relative densities of HA-VP3. The activity of binding of each Flag-VP6 mutant to HA-VP3 was calculated in five experiments, and the results (means ± SDs) are shown as a percentage of the binding of the WT. *, a significant difference in comparison to the binding activity of WT Flag-VP6 ( P

Techniques Used: Binding Assay, Mutagenesis, Sequencing, Immunoprecipitation, Activity Assay

Interaction of VP6 with VP3. (A) Copurification of VP6 with VP3, each of which was expressed using a baculovirus expression system. His-tagged VP6 (His-VP6) was coexpressed with either HA-tagged VP3 (HA-VP3) or nontagged VP3 (VP3) in Sf9 cells. In parallel, His-tagged VP3 (VP3) was coexpressed with nontagged VP6 (VP6). As a control, either His-VP6 or His-VP3 was expressed. Proteins were purified with a His-Select nickel affinity gel. (B) Analysis of the VP6/VP3 complex by gel filtration chromatography. Two types of VP3/VP6 complexes, His-VP6/HA-VP3 (green solid line) and His-VP6/VP3 (blue dashed line), copurified using nickel affinity gels, were loaded onto an equilibrated HiPrep 16/60 Sephacryl S-300 HR gel filtration column and eluted with the same buffer at a flow rate of 0.5 ml/min. His-VP6 (red dotted line) was loaded as a control. The left vertical axis indicates the absorbance at 280 nm of His-VP6 and the His-VP6/HA-VP3 complex. The right vertical axis indicates the absorbance at 280 nm of the His-VP6/VP3 complex. mAU, milli-absorbance units. (C) Fractions of the His-VP6/HA-VP3 complex (fractions 1 to 13) were collected between elution volumes of 38 ml and 77 ml and analyzed using SDS-PAGE (top). As a control, the same fractions of His-VP6 were analyzed (bottom).
Figure Legend Snippet: Interaction of VP6 with VP3. (A) Copurification of VP6 with VP3, each of which was expressed using a baculovirus expression system. His-tagged VP6 (His-VP6) was coexpressed with either HA-tagged VP3 (HA-VP3) or nontagged VP3 (VP3) in Sf9 cells. In parallel, His-tagged VP3 (VP3) was coexpressed with nontagged VP6 (VP6). As a control, either His-VP6 or His-VP3 was expressed. Proteins were purified with a His-Select nickel affinity gel. (B) Analysis of the VP6/VP3 complex by gel filtration chromatography. Two types of VP3/VP6 complexes, His-VP6/HA-VP3 (green solid line) and His-VP6/VP3 (blue dashed line), copurified using nickel affinity gels, were loaded onto an equilibrated HiPrep 16/60 Sephacryl S-300 HR gel filtration column and eluted with the same buffer at a flow rate of 0.5 ml/min. His-VP6 (red dotted line) was loaded as a control. The left vertical axis indicates the absorbance at 280 nm of His-VP6 and the His-VP6/HA-VP3 complex. The right vertical axis indicates the absorbance at 280 nm of the His-VP6/VP3 complex. mAU, milli-absorbance units. (C) Fractions of the His-VP6/HA-VP3 complex (fractions 1 to 13) were collected between elution volumes of 38 ml and 77 ml and analyzed using SDS-PAGE (top). As a control, the same fractions of His-VP6 were analyzed (bottom).

Techniques Used: Copurification, Expressing, Purification, Filtration, Chromatography, Flow Cytometry, SDS Page

Assay of VP6/VP3 interaction-defective BTV, BTV RYF/3A (RYF/3A), and BTV d278/287 (d278/287) replication. Each 100 μl (∼1 × 10 3 PFU) of VP6/VP3 interaction-defective BTV once amplified in BSR-VP6 cells was inoculated into either WT-BSR cells (light gray) or BSR-VP6 cells (dark gray). At 24 h postinoculation, the total virus titer (mean ± SD) was determined by plaque assay. As a control, cells were infected with BTV RA (RA).
Figure Legend Snippet: Assay of VP6/VP3 interaction-defective BTV, BTV RYF/3A (RYF/3A), and BTV d278/287 (d278/287) replication. Each 100 μl (∼1 × 10 3 PFU) of VP6/VP3 interaction-defective BTV once amplified in BSR-VP6 cells was inoculated into either WT-BSR cells (light gray) or BSR-VP6 cells (dark gray). At 24 h postinoculation, the total virus titer (mean ± SD) was determined by plaque assay. As a control, cells were infected with BTV RA (RA).

Techniques Used: Amplification, Plaque Assay, Infection

Direct interaction of VP6 with VP3 at a region between residues 279 and 286. (A) Coimmunoprecipitation of VP6 with VP3. HA-tagged VP3 (HA-VP3) was expressed in WT-BSR cells in the presence or absence of Flag-tagged VP6 (Flag-VP6). HA-VP3 and Flag-VP6 were immunoprecipitated using a mouse anti-HA MAb and a mouse anti-Flag MAb, respectively. Precipitated proteins were detected by immunoblotting using a rabbit anti-HA pAb and a rabbit anti-Flag pAb, respectively. (B) Schematic representation of modified VP6. Numbers in the middle column indicate amino acid positions in VP6 where changes were introduced. Note that no Flag tag was inserted at the N-terminal end of the three VP6 mutants N 87 C 115 , N 87 C 29 , and N 87 . +, coimmunoprecipitation; IP, immunoprecipitation; IB, immunoblotting; aa, amino acid; ND, not determined.
Figure Legend Snippet: Direct interaction of VP6 with VP3 at a region between residues 279 and 286. (A) Coimmunoprecipitation of VP6 with VP3. HA-tagged VP3 (HA-VP3) was expressed in WT-BSR cells in the presence or absence of Flag-tagged VP6 (Flag-VP6). HA-VP3 and Flag-VP6 were immunoprecipitated using a mouse anti-HA MAb and a mouse anti-Flag MAb, respectively. Precipitated proteins were detected by immunoblotting using a rabbit anti-HA pAb and a rabbit anti-Flag pAb, respectively. (B) Schematic representation of modified VP6. Numbers in the middle column indicate amino acid positions in VP6 where changes were introduced. Note that no Flag tag was inserted at the N-terminal end of the three VP6 mutants N 87 C 115 , N 87 C 29 , and N 87 . +, coimmunoprecipitation; IP, immunoprecipitation; IB, immunoblotting; aa, amino acid; ND, not determined.

Techniques Used: Immunoprecipitation, Modification, FLAG-tag

Nonrecruitment of VP6/VP3 interaction-defective VP6 as well as VP1, VP4, and RNAs into core particles purified from WT-BSR cells. (A) Electron microscopy of BTV RYF/3A core particles amplified in either BSR-VP6 cells (top) or normal BSR cells (bottom). Bars, 100 nm. (B) The incorporation of VP6 proteins with core particles was analyzed using immunoblotting (left). VP6 proteins were normalized with VP7, and the level of incorporation with VP6 into the particles is shown as a percentage of that for VP7 proteins (right). (C) Analysis of VP6/VP3 interaction-defective BTV core particles purified from WT-BSR and BSR-VP6 cells by CsCl gradient centrifugation. Supernatants from BSR-VP6 (left) or BSR (right) infected cell lysates were spun down over a 30% (wt/vol) sucrose cushion and subjected to CsCl equilibrium centrifugation. Thirteen fractions were collected from the top, and then 12 fractions from the top were analyzed by SDS-PAGE. The gels were stained with Coomassie brilliant blue.
Figure Legend Snippet: Nonrecruitment of VP6/VP3 interaction-defective VP6 as well as VP1, VP4, and RNAs into core particles purified from WT-BSR cells. (A) Electron microscopy of BTV RYF/3A core particles amplified in either BSR-VP6 cells (top) or normal BSR cells (bottom). Bars, 100 nm. (B) The incorporation of VP6 proteins with core particles was analyzed using immunoblotting (left). VP6 proteins were normalized with VP7, and the level of incorporation with VP6 into the particles is shown as a percentage of that for VP7 proteins (right). (C) Analysis of VP6/VP3 interaction-defective BTV core particles purified from WT-BSR and BSR-VP6 cells by CsCl gradient centrifugation. Supernatants from BSR-VP6 (left) or BSR (right) infected cell lysates were spun down over a 30% (wt/vol) sucrose cushion and subjected to CsCl equilibrium centrifugation. Thirteen fractions were collected from the top, and then 12 fractions from the top were analyzed by SDS-PAGE. The gels were stained with Coomassie brilliant blue.

Techniques Used: Purification, Electron Microscopy, Amplification, Gradient Centrifugation, Infection, Centrifugation, SDS Page, Staining

5) Product Images from "Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]"

Article Title: Scaffold Function of Ca2+-Dependent Protein Kinase: Tobacco Ca2+-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C]-DEPENDENT PROTEIN KINASE1 Transfers 14-3-3 to the Substrate REPRESSION OF SHOOT GROWTH after Phosphorylation 1 [C] [W]

Journal: Plant Physiology

doi: 10.1104/pp.114.236448

14-3-3 shows higher affinity for RSG than for NtCDPK1. A, Autophosphorylated MBP-NtCDPK1 and phosphorylated MBP-RSG were immobilized on amylose resin and incubated with His-14-3-3. NtCDPK1- or RSG-bound proteins were subjected to SDS-PAGE, followed by
Figure Legend Snippet: 14-3-3 shows higher affinity for RSG than for NtCDPK1. A, Autophosphorylated MBP-NtCDPK1 and phosphorylated MBP-RSG were immobilized on amylose resin and incubated with His-14-3-3. NtCDPK1- or RSG-bound proteins were subjected to SDS-PAGE, followed by

Techniques Used: Incubation, SDS Page

14-3-3 has no effect on the kinase activity of NtCDPK1. Autophosphorylated GST-NtCDPK1 was immobilized on glutathione beads and incubated with or without His-14-3-3 in the presence of Ca 2+ . Glutathione beads into which GST-NtCDPK1 and His-14-3-3 had been
Figure Legend Snippet: 14-3-3 has no effect on the kinase activity of NtCDPK1. Autophosphorylated GST-NtCDPK1 was immobilized on glutathione beads and incubated with or without His-14-3-3 in the presence of Ca 2+ . Glutathione beads into which GST-NtCDPK1 and His-14-3-3 had been

Techniques Used: Activity Assay, Incubation

NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads
Figure Legend Snippet: NtCDPK1 forms a heterotrimer with RSG and 14-3-3. A, RSG binds to the complex of NtCDPK1 with 14-3-3. GST-NtCDPK1 was autophosphorylated and absorbed into glutathione beads. The immobilized GST-NtCDPK1 was incubated with His-14-3-3. Glutathione beads

Techniques Used: Incubation

14-3-3 interacts with NtCDPK1. A, 14-3-3 interacts with NtCDPK1 in an autophosphorylation- and Ca 2+ -dependent manner. GST and nonphosphorylated or autophosphorylated GST-NtCDPK1 were immobilized on glutathione beads and incubated with His-14-3-3 in the
Figure Legend Snippet: 14-3-3 interacts with NtCDPK1. A, 14-3-3 interacts with NtCDPK1 in an autophosphorylation- and Ca 2+ -dependent manner. GST and nonphosphorylated or autophosphorylated GST-NtCDPK1 were immobilized on glutathione beads and incubated with His-14-3-3 in the

Techniques Used: Incubation

6) Product Images from "Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies"

Article Title: Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.12.006

SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
Figure Legend Snippet: SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).

Techniques Used: SDS Page, Purification, Molecular Weight, Marker

7) Product Images from "Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana"

Article Title: Calcium signalling regulates the functions of the bZIP protein VIP1 in touch responses in Arabidopsis thaliana

Journal: Annals of Botany

doi: 10.1093/aob/mcy125

Hexahistidine-tagged CAM1 (His-CAM1) interacts with GST-fused group I bZIP proteins and their derivatives in vitro . His-CAM1 and GST-fused proteins were expressed in Escherichia coli , purified, and used for in vitro crosslink assays. For each panel, the
Figure Legend Snippet: Hexahistidine-tagged CAM1 (His-CAM1) interacts with GST-fused group I bZIP proteins and their derivatives in vitro . His-CAM1 and GST-fused proteins were expressed in Escherichia coli , purified, and used for in vitro crosslink assays. For each panel, the

Techniques Used: In Vitro, Purification

8) Product Images from "Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity"

Article Title: Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity

Journal: Journal of Virology

doi: 10.1128/JVI.01784-17

Localization of VP6, VP3, and NS2 in BTV-infected cells. WT-BSR cells were infected with each of WT-BTV (A), RA-BTV (B), RYF/3A-BTV (C), and d278/287-BTV (D), expressing WT VP6, RA VP6, RYF/3A VP6, and d278/287 VP6, respectively. Note that the cells were infected with WT-BTV and RA-BTV at an MOI of 0.1. At 24 h postinfection, the expression of VP6, VP3, and NS2 was observed using confocal microscopy. VP6 was detected using a guinea pig anti-VP6 antibody (top) and a rabbit anti-VP6 antibody (middle). VP3 was detected using a mouse anti-VP3 antibody. NS2 was detected using a guinea pig anti-NS2 antibody.
Figure Legend Snippet: Localization of VP6, VP3, and NS2 in BTV-infected cells. WT-BSR cells were infected with each of WT-BTV (A), RA-BTV (B), RYF/3A-BTV (C), and d278/287-BTV (D), expressing WT VP6, RA VP6, RYF/3A VP6, and d278/287 VP6, respectively. Note that the cells were infected with WT-BTV and RA-BTV at an MOI of 0.1. At 24 h postinfection, the expression of VP6, VP3, and NS2 was observed using confocal microscopy. VP6 was detected using a guinea pig anti-VP6 antibody (top) and a rabbit anti-VP6 antibody (middle). VP3 was detected using a mouse anti-VP3 antibody. NS2 was detected using a guinea pig anti-NS2 antibody.

Techniques Used: Infection, Expressing, Confocal Microscopy

Localization of chimeric VP6-EGFP proteins in WT-BSR cells infected with various chimeric BTV strains. (A) Schematic representation of the changes introduced in BTV VP6. The name of the mutation is indicated on the left. Numbers in the middle column indicate amino acid (aa) positions in VP6, where EGFPs were fused with the N- and/or C-terminal region of VP6. +, colocalization of VP6-EGFP with NS2. (B) Colocalization of VP6 to which EGFP was fused at the C terminus with NS2. Either BTV/N 87 (top) or BTV/N 87 C 115 (bottom) was used to infect WT-BSR cells at an MOI of 1.0. At 24 h postinfection, the expression of EGFP and NS2 was observed using confocal microscopy. NS2 was detected using an anti-NS2 antibody produced in a guinea pig. Open and closed arrowheads, punctate structures of VP6 and VIBs, respectively.
Figure Legend Snippet: Localization of chimeric VP6-EGFP proteins in WT-BSR cells infected with various chimeric BTV strains. (A) Schematic representation of the changes introduced in BTV VP6. The name of the mutation is indicated on the left. Numbers in the middle column indicate amino acid (aa) positions in VP6, where EGFPs were fused with the N- and/or C-terminal region of VP6. +, colocalization of VP6-EGFP with NS2. (B) Colocalization of VP6 to which EGFP was fused at the C terminus with NS2. Either BTV/N 87 (top) or BTV/N 87 C 115 (bottom) was used to infect WT-BSR cells at an MOI of 1.0. At 24 h postinfection, the expression of EGFP and NS2 was observed using confocal microscopy. NS2 was detected using an anti-NS2 antibody produced in a guinea pig. Open and closed arrowheads, punctate structures of VP6 and VIBs, respectively.

Techniques Used: Infection, Mutagenesis, Expressing, Confocal Microscopy, Produced

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Electrophoresis:

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Article Snippet: .. Tris-HCl, EDTA and MgCl2 for electrophoresis analysis were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). .. Water was deionized (18.0 MΩ cm specific resistance) by a Milli-Q system (Millipore Corp., Bedford, MA).

Incubation:

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Article Title: Methylation of Xenopus CIRP2 regulates its arginine- and glycine-rich region-mediated nucleocytoplasmic distribution
Article Snippet: .. In order to examine the interaction between xCIRP2 and xPRMT1 in vitro , 1 µg GST or GST-xCIRP2 was incubated with 2 µg of 6× histidine-tagged xPRMT1 in 100 µl of reaction mixture containing 20 mM Tris–HCl (pH 7.5) and 100 mM NaCl with or without 80 µM S -adenosyl- l -methionine (AdoMet; Nacalai Tesque, Kyoto, Japan) at 37°C for 60 min. Glutathione–Sepharose beads were then added into the reaction mixture and the mixture was incubated at 4°C for 60 min, following which the beads were washed four times in wash buffer [20 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethanesulfonyl fluoride (PMSF)]. .. After removing the final wash, the samples were analyzed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and the gel was stained with silver.

other:

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In Vitro:

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Infection:

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    Nacalai cosmogel his accept column
    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a <t>COSMOGEL</t> His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
    Cosmogel His Accept Column, supplied by Nacalai, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Nacalai cosmogel his accept
    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a <t>COSMOGEL</t> His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
    Cosmogel His Accept, supplied by Nacalai, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cosmogel his accept/product/Nacalai
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    Price from $9.99 to $1999.99
    cosmogel his accept - by Bioz Stars, 2020-08
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    91
    Nacalai ni agarose
    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a <t>COSMOGEL</t> His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).
    Ni Agarose, supplied by Nacalai, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).

    Journal: Biochemistry and Biophysics Reports

    Article Title: Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

    doi: 10.1016/j.bbrep.2016.12.006

    Figure Lengend Snippet: SDS-PAGE analysis of the protein fractions collected at each step of purification of HBx(101−136)-(GS) 5 -Bcl-x L . Lane 1, molecular weight marker; lane 2, supernatant; lane 3, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L purified by a COSMOGEL His-accept column; lane 4, His 6 -HBx(101−136)-(GS) 5 -Bcl-x L after dialysis; lane 5, HBx(101−136)-(GS) 5 -Bcl-x L after cleavage of the His 6 -tag by thrombin; lane 6, HBx(101−136)-(GS) 5 -Bcl-x L purified by a Resource Q anion exchange column; lanes 7–9, purified HBx(101−136)-(GS) 5 -Bcl-x L (1-, 3- and 5-µg loadings, respectively).

    Article Snippet: The supernatant containing the His6 -tagged protein, which was obtained by centrifugation, was loaded onto a COSMOGEL His-accept column (3–4 mL bed volume) (Nacalai Tesque Inc.).

    Techniques: SDS Page, Purification, Molecular Weight, Marker