Structured Review

GE Healthcare h10 t mtc28 bap protein
Purification of <t>MTC28-BAP</t> protein. Chromatogram showing (A) Elution profile of <t>H10-T-MTC28-BAP</t> protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on Superdex 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.
H10 T Mtc28 Bap Protein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 1837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection"

Article Title: Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191315

Purification of MTC28-BAP protein. Chromatogram showing (A) Elution profile of H10-T-MTC28-BAP protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on Superdex 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.
Figure Legend Snippet: Purification of MTC28-BAP protein. Chromatogram showing (A) Elution profile of H10-T-MTC28-BAP protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on Superdex 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.

Techniques Used: Purification, Flow Cytometry, Affinity Column, Filtration, Affinity Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Marker, Homogenization

2) Product Images from "The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex"

Article Title: The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200210028

The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1 , and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl ® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.
Figure Legend Snippet: The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1 , and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl ® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.

Techniques Used: Purification, Translocation Assay, In Vitro, Activity Assay, Chromatography, Mass Spectrometry, Staining, SDS Page

3) Product Images from "One-Step Purification of Enterocytozoon bieneusi Spores from Human Stools by Immunoaffinity Expanded-Bed Adsorption"

Article Title: One-Step Purification of Enterocytozoon bieneusi Spores from Human Stools by Immunoaffinity Expanded-Bed Adsorption

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.39.5.1947-1951.2001

Chromatogram of spore purification using the immuno-Streamline rProtein A matrix. Features: column, Streamline 25 (75 ml of gel); sample, 75 ml of the spore suspension; buffer, PBS at pH 7.2; detection at 280 nm ( y axis); elution volume ( x axis); flow rate, 2 ml/min in equilibration and injection (I), 32 ml/min in expansion (Ex), and 15 ml/min in sedimented bed for elution (El). The fractions between 500 and 1,500 ml of elution volume represent fecal debris eliminated after expansion. The peak at ca. 1,600 ml of elution volume represents the eluted spores.
Figure Legend Snippet: Chromatogram of spore purification using the immuno-Streamline rProtein A matrix. Features: column, Streamline 25 (75 ml of gel); sample, 75 ml of the spore suspension; buffer, PBS at pH 7.2; detection at 280 nm ( y axis); elution volume ( x axis); flow rate, 2 ml/min in equilibration and injection (I), 32 ml/min in expansion (Ex), and 15 ml/min in sedimented bed for elution (El). The fractions between 500 and 1,500 ml of elution volume represent fecal debris eliminated after expansion. The peak at ca. 1,600 ml of elution volume represents the eluted spores.

Techniques Used: Purification, Flow Cytometry, Injection

4) Product Images from "Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response"

Article Title: Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00754-12

Opsonophagocytic assay. Pneumococcal strain 679/99 (PspA clade 3, serotype 6B, used to test the opsonic activity of anti-PS6B antibodies [Ab]) (A) and pneumococcal strain 245/00 (PspA clade 1, serotype 14, used to test the opsonic activity of anti-PspA1 antibodies) (B) were incubated with the sera from mice immunized with PS6B-rPspA1 or with PS6B-OCT-mPspA1 and a complement source. The opsonized pneumococci were incubated with peritoneal cells and plated on blood agar plates. Sera from mice immunized with saline plus Al(OH) 3 or with PS6B coadministered with rPspA1 or mPspA1 were used as controls. The numbers of CFU recovered after 20 h were compared by one-way ANOVA with Tukey's multiple-comparison test. The lines on the graph represent means. Asterisks indicate statistically significant differences (**, P
Figure Legend Snippet: Opsonophagocytic assay. Pneumococcal strain 679/99 (PspA clade 3, serotype 6B, used to test the opsonic activity of anti-PS6B antibodies [Ab]) (A) and pneumococcal strain 245/00 (PspA clade 1, serotype 14, used to test the opsonic activity of anti-PspA1 antibodies) (B) were incubated with the sera from mice immunized with PS6B-rPspA1 or with PS6B-OCT-mPspA1 and a complement source. The opsonized pneumococci were incubated with peritoneal cells and plated on blood agar plates. Sera from mice immunized with saline plus Al(OH) 3 or with PS6B coadministered with rPspA1 or mPspA1 were used as controls. The numbers of CFU recovered after 20 h were compared by one-way ANOVA with Tukey's multiple-comparison test. The lines on the graph represent means. Asterisks indicate statistically significant differences (**, P

Techniques Used: Opsonophagocytic Assay, Activity Assay, Incubation, Mouse Assay

5) Product Images from "Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography"

Article Title: Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography

Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

doi: 10.1016/j.jchromb.2008.02.014

Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.
Figure Legend Snippet: Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.

Techniques Used: Purification, Injection, Generated

Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.
Figure Legend Snippet: Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.

Techniques Used: Purification, Injection, Generated

6) Product Images from "Development of novel antibodies for detection of mobile colistin-resistant bacteria contaminated in meats"

Article Title: Development of novel antibodies for detection of mobile colistin-resistant bacteria contaminated in meats

Journal: Scientific Reports

doi: 10.1038/s41598-018-34764-2

Cropped Western blot of mAb reactivity to MCR-1 and MCR-2. Lane 1, Protein markers with molecular weights (kDa) indicated at the left; Lane 2, Purified cMCR-1 with His-tag removed (0.2 µg); Lane 3, Purified cMCR-2 with His-tag on (0.2 µg); Lanes 4 and 5, Purified His-full-length MCR-1 and His-full-length MCR-2. Blots were probed with indicated mAbs following SDS-PAGE under non-reducing condition. The expected sizes MCR-1 and MCR-2 proteins are indicated at the right side of the blot. Full-length blots are presented in Supplementary Fig. S3 .
Figure Legend Snippet: Cropped Western blot of mAb reactivity to MCR-1 and MCR-2. Lane 1, Protein markers with molecular weights (kDa) indicated at the left; Lane 2, Purified cMCR-1 with His-tag removed (0.2 µg); Lane 3, Purified cMCR-2 with His-tag on (0.2 µg); Lanes 4 and 5, Purified His-full-length MCR-1 and His-full-length MCR-2. Blots were probed with indicated mAbs following SDS-PAGE under non-reducing condition. The expected sizes MCR-1 and MCR-2 proteins are indicated at the right side of the blot. Full-length blots are presented in Supplementary Fig. S3 .

Techniques Used: Western Blot, Purification, SDS Page

Standard curves for ELISA detection of cMCR-1 ( a ) and cMCR-2 ( b ) in PBS. Luminescence counts (cps) were plotted against the concentration of MCR-1 and MCR-2 proteins (ng/mL). Data represent means of triplicate samples ± SD. The dashed lines in the inset are the upper and lower 95% confidence limits of the linear regression line.
Figure Legend Snippet: Standard curves for ELISA detection of cMCR-1 ( a ) and cMCR-2 ( b ) in PBS. Luminescence counts (cps) were plotted against the concentration of MCR-1 and MCR-2 proteins (ng/mL). Data represent means of triplicate samples ± SD. The dashed lines in the inset are the upper and lower 95% confidence limits of the linear regression line.

Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay

Analysis of His-tagged recombinant MCR-1 and MCR-2 proteins. ( a ) Cropped SDS-PAGE of partially purified MCR recombinant proteins stained with SimplyBlue: lane 1, Protein markers with molecular weights (kDa) indicated at the left; lane 2, cMCR-1; lane 3, cMCR-2; lane 4, MCR-1; lane 5, MCR-2. Full-length gel is presented in Supplementary Fig. S1a . ( b ) Cropped Western blot of partially purified MCR recombinant proteins using anti-His antibody: lane 1, Protein markers with molecular weights (kDa) indicated at the left; lane 2, cMCR-1; lane 3, cMCR-2; lane 4, MCR-1; lane 5, MCR-2. The predicted His-MCR (full-length) and His-cMCR (catalytic domain) protein bands are indicated by arrows at the right. Full-length blot is presented in Supplementary Fig. S1b . ( c ) MS verification of the N-terminal His-tagged MCR-1 full-length protein. The tryptic peptides (underlined) and the chymotryptic peptides (highlighted in yellow) cover 48.6% and 42.5% of the MCR-1 sequence, respectively. The overall coverage from tryptic and chymotryptic peptides was 60.6%.
Figure Legend Snippet: Analysis of His-tagged recombinant MCR-1 and MCR-2 proteins. ( a ) Cropped SDS-PAGE of partially purified MCR recombinant proteins stained with SimplyBlue: lane 1, Protein markers with molecular weights (kDa) indicated at the left; lane 2, cMCR-1; lane 3, cMCR-2; lane 4, MCR-1; lane 5, MCR-2. Full-length gel is presented in Supplementary Fig. S1a . ( b ) Cropped Western blot of partially purified MCR recombinant proteins using anti-His antibody: lane 1, Protein markers with molecular weights (kDa) indicated at the left; lane 2, cMCR-1; lane 3, cMCR-2; lane 4, MCR-1; lane 5, MCR-2. The predicted His-MCR (full-length) and His-cMCR (catalytic domain) protein bands are indicated by arrows at the right. Full-length blot is presented in Supplementary Fig. S1b . ( c ) MS verification of the N-terminal His-tagged MCR-1 full-length protein. The tryptic peptides (underlined) and the chymotryptic peptides (highlighted in yellow) cover 48.6% and 42.5% of the MCR-1 sequence, respectively. The overall coverage from tryptic and chymotryptic peptides was 60.6%.

Techniques Used: Recombinant, SDS Page, Purification, Staining, Western Blot, Mass Spectrometry, Sequencing

7) Product Images from "Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection"

Article Title: Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191315

Purification of MTC28-BAP protein. Chromatogram showing (A) Elution profile of H10-T-MTC28-BAP protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on Superdex 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.
Figure Legend Snippet: Purification of MTC28-BAP protein. Chromatogram showing (A) Elution profile of H10-T-MTC28-BAP protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on Superdex 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.

Techniques Used: Purification, Flow Cytometry, Affinity Column, Filtration, Affinity Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Marker, Homogenization

8) Product Images from "Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography"

Article Title: Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography

Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

doi: 10.1016/j.jchromb.2008.02.014

Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.
Figure Legend Snippet: Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.

Techniques Used: Purification, Injection, Generated

Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.
Figure Legend Snippet: Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.

Techniques Used: Purification, Injection, Generated

9) Product Images from "Transepithelial Transport of PAMAM Dendrimers across Isolated Rat Jejunal Mucosae in Ussing Chambers"

Article Title: Transepithelial Transport of PAMAM Dendrimers across Isolated Rat Jejunal Mucosae in Ussing Chambers

Journal: Biomacromolecules

doi: 10.1021/bm5004465

Percent TEER changes of isolated jejunal tissue: control (●), G4 dendrimers 1.0 mM (■), G3.5 dendrimers 1.0 mM (◆), FITC-dextran 4 kDa (Δ), FITC-dextran 10 kDa (∇). Percent TEER values were calculated as a percentage of the initial TEER at t = 0 in each group. Significant differences from TEER of each individual group at t = 0 are marked with open circles (G3.5 dendrimers), star (control, G3.5 dendrimers, FITC-dextran 4 kDa), and asterisk (all groups).
Figure Legend Snippet: Percent TEER changes of isolated jejunal tissue: control (●), G4 dendrimers 1.0 mM (■), G3.5 dendrimers 1.0 mM (◆), FITC-dextran 4 kDa (Δ), FITC-dextran 10 kDa (∇). Percent TEER values were calculated as a percentage of the initial TEER at t = 0 in each group. Significant differences from TEER of each individual group at t = 0 are marked with open circles (G3.5 dendrimers), star (control, G3.5 dendrimers, FITC-dextran 4 kDa), and asterisk (all groups).

Techniques Used: Isolation

P app of FITC-PAMAM (1.0 mM), FITC-dextran 4 kDa (0.625 mM), FITC-dextran 10 kDa (0.25 mM), and free FITC (0.02 mM) across isolated rat jejunum. FITC-G3.5 dendrimers had significantly increased P app compared to free FITC (* p
Figure Legend Snippet: P app of FITC-PAMAM (1.0 mM), FITC-dextran 4 kDa (0.625 mM), FITC-dextran 10 kDa (0.25 mM), and free FITC (0.02 mM) across isolated rat jejunum. FITC-G3.5 dendrimers had significantly increased P app compared to free FITC (* p

Techniques Used: Isolation

P app of [ 14 C]-mannitol through isolated rat jejunum. No significant difference was observed for G4.0 and G3.5 dendrimer (1.0 mM) treatments vs free FITC, indicating no enhanced paracellular transport in the presence of either dendrimer.
Figure Legend Snippet: P app of [ 14 C]-mannitol through isolated rat jejunum. No significant difference was observed for G4.0 and G3.5 dendrimer (1.0 mM) treatments vs free FITC, indicating no enhanced paracellular transport in the presence of either dendrimer.

Techniques Used: Isolation

Δ I SC response to basolateral additions of carbachol to jejunal mucosae. No significant difference in response was observed for test groups: control (●), G4 dendrimers 1.0 mM (■), G3.5 dendrimers 1.0 mM (◆), FITC-dextran 4 kDa (Δ), FITC-dextran 10 kDa (∇).
Figure Legend Snippet: Δ I SC response to basolateral additions of carbachol to jejunal mucosae. No significant difference in response was observed for test groups: control (●), G4 dendrimers 1.0 mM (■), G3.5 dendrimers 1.0 mM (◆), FITC-dextran 4 kDa (Δ), FITC-dextran 10 kDa (∇).

Techniques Used:

10) Product Images from "The molecular mechanism of flop-selectivity and subsite recognition for an AMPA receptor allosteric modulator: Structures of GluA2 and GluA3 complexed with PEPA"

Article Title: The molecular mechanism of flop-selectivity and subsite recognition for an AMPA receptor allosteric modulator: Structures of GluA2 and GluA3 complexed with PEPA

Journal: Biochemistry

doi: 10.1021/bi1000678

(A) Comparison of glutamate-bound GluA3 o S1S2 in the presence (blue) and the absence of PEPA (green) in two orientations. Both orientations of PEPA are shown. Note that the binding of PEPA results in a separation of the two components of the dimer (distance between the Cα atoms of the threonine in the linker) by approximately 2.5 Å. (B) One monomer of GluA3 o S1S2 in the presence (blue) and the absence of PEPA (green) with one orientation of PEPA shown. Shown for comparison is the PEPA-bound form of GluA2 o (red). Both the J/K helices and the strand near S497 are displaced upon binding PEPA for both GluA2 o and GluA3 o . Also, the sidechains of S497 and S729 are in different rotameric states for GluA3 o bound to PEPA compared with GluA3 o in the absence of PEPA and GluA2 o bound to PEPA. Also, N754 is displaced in PEPA-bound GluA3 o , such that only one H-bond is possible with the amide of PEPA.
Figure Legend Snippet: (A) Comparison of glutamate-bound GluA3 o S1S2 in the presence (blue) and the absence of PEPA (green) in two orientations. Both orientations of PEPA are shown. Note that the binding of PEPA results in a separation of the two components of the dimer (distance between the Cα atoms of the threonine in the linker) by approximately 2.5 Å. (B) One monomer of GluA3 o S1S2 in the presence (blue) and the absence of PEPA (green) with one orientation of PEPA shown. Shown for comparison is the PEPA-bound form of GluA2 o (red). Both the J/K helices and the strand near S497 are displaced upon binding PEPA for both GluA2 o and GluA3 o . Also, the sidechains of S497 and S729 are in different rotameric states for GluA3 o bound to PEPA compared with GluA3 o in the absence of PEPA and GluA2 o bound to PEPA. Also, N754 is displaced in PEPA-bound GluA3 o , such that only one H-bond is possible with the amide of PEPA.

Techniques Used: Binding Assay

The PEPA binding site, emphasizing the important interactions, shown in two orientations. (A) A view of the amide side of PEPA bound to GluA2 S1S2. The hydrogen bonding network with the amide of PEPA is shown as dotted lines. The H-bond with the sidechain of S729 is difficult to display in the orientation used in the figure. (B) A view of the phenyl group of PEPA inserted into a hydrophobic pocket in GluA2 S1S2. (C) RMS plot showing more variability in the J/K helices for the PEPA-bound structure than the unbound structure. (D) J/K helix showing where differences in the two orientations were analyzed. The amide of PEPA-N754 interaction (blue) maintains the position of the J helix in the absence of PEPA (green) The J helix is displaced on the phenyl side of PEPA (red).
Figure Legend Snippet: The PEPA binding site, emphasizing the important interactions, shown in two orientations. (A) A view of the amide side of PEPA bound to GluA2 S1S2. The hydrogen bonding network with the amide of PEPA is shown as dotted lines. The H-bond with the sidechain of S729 is difficult to display in the orientation used in the figure. (B) A view of the phenyl group of PEPA inserted into a hydrophobic pocket in GluA2 S1S2. (C) RMS plot showing more variability in the J/K helices for the PEPA-bound structure than the unbound structure. (D) J/K helix showing where differences in the two orientations were analyzed. The amide of PEPA-N754 interaction (blue) maintains the position of the J helix in the absence of PEPA (green) The J helix is displaced on the phenyl side of PEPA (red).

Techniques Used: Binding Assay

11) Product Images from "Purification, crystallization and phase determination of the DR1998 haem b catalase from Deinococcus radiodurans"

Article Title: Purification, crystallization and phase determination of the DR1998 haem b catalase from Deinococcus radiodurans

Journal: Acta Crystallographica. Section F, Structural Biology Communications

doi: 10.1107/S2053230X1400764X

( a ) SDS–PAGE. Lane 1, low-molecular-weight markers (kDa); lane 2, DR1998 catalase protein eluted from HiTrap Q-HP. ( b ) UV–Vis absorption spectrum of DR1998 catalase indicating the presence of a  b -type haem.
Figure Legend Snippet: ( a ) SDS–PAGE. Lane 1, low-molecular-weight markers (kDa); lane 2, DR1998 catalase protein eluted from HiTrap Q-HP. ( b ) UV–Vis absorption spectrum of DR1998 catalase indicating the presence of a b -type haem.

Techniques Used: SDS Page, Molecular Weight

12) Product Images from "Purification, crystallization and phase determination of the DR1998 haem b catalase from Deinococcus radiodurans"

Article Title: Purification, crystallization and phase determination of the DR1998 haem b catalase from Deinococcus radiodurans

Journal: Acta Crystallographica. Section F, Structural Biology Communications

doi: 10.1107/S2053230X1400764X

Solvent-accessible molecular-surface representation of the molecular-replacement solution: A , B , C and D (in darker colours) correspond to the DR1998 asymmetric unit and A ′, B ′, C ′ and D ′ (in lighter colours) correspond to the crystallographic image from the operation (− x , y , − z + 1/2). This symmetry operation builds up the typical catalase oligomers ( ABA ′ B ′) and ( CDC ′ D ′), each with 222 point-group symmetry, upon combination of the crystallographic C 2 symmetry with local twofold symmetry axes. Directions x and y are in the plane of the paper and direction z is perpendicular to this plane.
Figure Legend Snippet: Solvent-accessible molecular-surface representation of the molecular-replacement solution: A , B , C and D (in darker colours) correspond to the DR1998 asymmetric unit and A ′, B ′, C ′ and D ′ (in lighter colours) correspond to the crystallographic image from the operation (− x , y , − z + 1/2). This symmetry operation builds up the typical catalase oligomers ( ABA ′ B ′) and ( CDC ′ D ′), each with 222 point-group symmetry, upon combination of the crystallographic C 2 symmetry with local twofold symmetry axes. Directions x and y are in the plane of the paper and direction z is perpendicular to this plane.

Techniques Used:

( a ) SDS–PAGE. Lane 1, low-molecular-weight markers (kDa); lane 2, DR1998 catalase protein eluted from HiTrap Q-HP. ( b ) UV–Vis absorption spectrum of DR1998 catalase indicating the presence of a b -type haem.
Figure Legend Snippet: ( a ) SDS–PAGE. Lane 1, low-molecular-weight markers (kDa); lane 2, DR1998 catalase protein eluted from HiTrap Q-HP. ( b ) UV–Vis absorption spectrum of DR1998 catalase indicating the presence of a b -type haem.

Techniques Used: SDS Page, Molecular Weight

13) Product Images from "Preparation of Recombinant Viral Glycoproteins for Novel and Therapeutic Antibody Discovery"

Article Title: Preparation of Recombinant Viral Glycoproteins for Novel and Therapeutic Antibody Discovery

Journal: Methods in Molecular Biology (Clifton, N.j.)

doi: 10.1007/978-1-59745-554-1_2

Transient expression of various sF constructs. 293 T cells were transfected with various sF constructs in the promoter modified pcDNA 3.1 Hygro (+) vector. Cell lysate and supernatant were precipitated with S-protein agarose. Western blot detection was
Figure Legend Snippet: Transient expression of various sF constructs. 293 T cells were transfected with various sF constructs in the promoter modified pcDNA 3.1 Hygro (+) vector. Cell lysate and supernatant were precipitated with S-protein agarose. Western blot detection was

Techniques Used: Expressing, Construct, Transfection, Modification, Plasmid Preparation, Western Blot

SDS-PAGE followed by coomassie stain analysis of S-protein agarose purified HeV and NiV sF GCN. 0.5 μL and 1.0 μL of protein purified from DMEM supplemented by 10% calf serum (DMEM-10) and reduced serum medium (OptiMEM) was analyzed on
Figure Legend Snippet: SDS-PAGE followed by coomassie stain analysis of S-protein agarose purified HeV and NiV sF GCN. 0.5 μL and 1.0 μL of protein purified from DMEM supplemented by 10% calf serum (DMEM-10) and reduced serum medium (OptiMEM) was analyzed on

Techniques Used: SDS Page, Staining, Purification

14) Product Images from "The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis"

Article Title: The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00583-18

The σAN-helix changes position within the RNAP. (Top left) The N-terminal α-helix (red) of σA (amino acids [aa] 149 to 162) is located within the primary channel of the M. smegmatis ) is positioned on the periphery in between the β and σ′ subunit claws, parallel to the NCD β′i1 finger. (Top right) σ701.1 from E. coli ). RNAP subunits are colored as follows: β, yellow; β′, green; α, gray; α′, cyan; ω, orange; σA/70, magenta; the N-terminal part of σA/70, dark blue; σAN-helix, red. (Bottom row) Detailed views of the respective σA/70N helix regions from the structures in the upper row.
Figure Legend Snippet: The σAN-helix changes position within the RNAP. (Top left) The N-terminal α-helix (red) of σA (amino acids [aa] 149 to 162) is located within the primary channel of the M. smegmatis ) is positioned on the periphery in between the β and σ′ subunit claws, parallel to the NCD β′i1 finger. (Top right) σ701.1 from E. coli ). RNAP subunits are colored as follows: β, yellow; β′, green; α, gray; α′, cyan; ω, orange; σA/70, magenta; the N-terminal part of σA/70, dark blue; σAN-helix, red. (Bottom row) Detailed views of the respective σA/70N helix regions from the structures in the upper row.

Techniques Used:

15) Product Images from "A diarylamine derived from anthranilic acid inhibits ZIKV replication"

Article Title: A diarylamine derived from anthranilic acid inhibits ZIKV replication

Journal: Scientific Reports

doi: 10.1038/s41598-019-54169-z

Analysis of FAM E3 intercalation into the viral dsRNA and its interaction with the activity of phage SP6 RNA polymerase. Fifteen nanomoles of dsRNA were incubated with the FAM E3 or intercalating controls (DMSO) or (DOX) for 45 minutes at room temperature. The reaction products were subjected to 1% agarose electrophoresis gel containing Ethidium Bromide followed by densitometry analysis ( a ). FAM E3 and 5 µg of purified pCCI-SP6-ZIKV amplicon was used for in vitro transcription using SP6 RNA polymerase at the presence or absence of FAM E3. Reaction products were analysed by agarose gel electrophoresis followed by densitometry analysis ( b ). Results of a representative of three independent reproducible experiments are shown.
Figure Legend Snippet: Analysis of FAM E3 intercalation into the viral dsRNA and its interaction with the activity of phage SP6 RNA polymerase. Fifteen nanomoles of dsRNA were incubated with the FAM E3 or intercalating controls (DMSO) or (DOX) for 45 minutes at room temperature. The reaction products were subjected to 1% agarose electrophoresis gel containing Ethidium Bromide followed by densitometry analysis ( a ). FAM E3 and 5 µg of purified pCCI-SP6-ZIKV amplicon was used for in vitro transcription using SP6 RNA polymerase at the presence or absence of FAM E3. Reaction products were analysed by agarose gel electrophoresis followed by densitometry analysis ( b ). Results of a representative of three independent reproducible experiments are shown.

Techniques Used: Activity Assay, Incubation, Electrophoresis, Purification, Amplification, In Vitro, Agarose Gel Electrophoresis

16) Product Images from "A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid"

Article Title: A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid

Journal: BioMed Research International

doi: 10.1155/2015/508656

ATR FT-IR spectra of GY785 DR (a) and GY785 DRS sulfated in BMImCl medium (b).
Figure Legend Snippet: ATR FT-IR spectra of GY785 DR (a) and GY785 DRS sulfated in BMImCl medium (b).

Techniques Used:

1 H NMR spectra of GY785 DR (a) and GY785 DRS (a′) and 13 C J-modulated NMR spectra of GY785 DR (b) and GY785 DRS (b′).
Figure Legend Snippet: 1 H NMR spectra of GY785 DR (a) and GY785 DRS (a′) and 13 C J-modulated NMR spectra of GY785 DR (b) and GY785 DRS (b′).

Techniques Used: Nuclear Magnetic Resonance

Comparison of the variation of the molecular weight and the sulfur content in GY785 DRS recovered following different sulfation time.
Figure Legend Snippet: Comparison of the variation of the molecular weight and the sulfur content in GY785 DRS recovered following different sulfation time.

Techniques Used: Molecular Weight

Cumulative molecular weight fraction of GY785 DRS with the dissolution step performed under atmospheric pressure (a) or under vacuum (100 mbar) (b).
Figure Legend Snippet: Cumulative molecular weight fraction of GY785 DRS with the dissolution step performed under atmospheric pressure (a) or under vacuum (100 mbar) (b).

Techniques Used: Molecular Weight

17) Product Images from "Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab"

Article Title: Heavy chain dimers stabilized by disulfide bonds are required to promote in vitro assembly of trastuzumab

Journal: BMC Molecular and Cell Biology

doi: 10.1186/s12860-019-0244-x

Graphical conclusions. Antibody folding is a complex process which occurs in vivo and where light chains are coupled to previously assembled heavy chain dimers. In this work, denaturation and chains separation of the Trastuzumab antibody followed by their in vitro refolding through slow dialysis method has shown that heavy chain dimers stabilized by disulphide bridges are necessary in order to reassemble the whole antibody. Successful in vitro assembly of heavy and light chains has been achieved when the chains have been independently produced
Figure Legend Snippet: Graphical conclusions. Antibody folding is a complex process which occurs in vivo and where light chains are coupled to previously assembled heavy chain dimers. In this work, denaturation and chains separation of the Trastuzumab antibody followed by their in vitro refolding through slow dialysis method has shown that heavy chain dimers stabilized by disulphide bridges are necessary in order to reassemble the whole antibody. Successful in vitro assembly of heavy and light chains has been achieved when the chains have been independently produced

Techniques Used: In Vivo, In Vitro, Produced

18) Product Images from "Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli"

Article Title: Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

doi: 10.1002/0471140864.ps0603s70

Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).
Figure Legend Snippet: Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).

Techniques Used: Filtration, Flow Cytometry, Purification, SDS Page, Migration

Superdex 200 chromatography in guanidine·HCl of SIV gp41 27-149 . SDS-PAGE of the numbered fractions is shown in the first inset (upper left); lane “a” contains molecular weight standards (bottom to top: 6.5, 14.4, 21.5, 31, 45, 66.2 kDa), and the purified protein migrates close to the 14.4 kDa standard. Lane “b” represents starting material loaded to column corresponding to guanidine·HCl-extracted inclusion bodies. Protein in the main peak (fractions 5 to 7) marked with arrow was used for protein folding after removal of guanidine·HCl by reversed-phase chromatography. The second inset (upper middle) refers to protein expression in minimal medium; lane A contains the same molecular weight standards as lane “a” in the first inset; lanes B and C correspond to insoluble protein and purified protein, respectively. Protein is labeled with 15 N and 13 .
Figure Legend Snippet: Superdex 200 chromatography in guanidine·HCl of SIV gp41 27-149 . SDS-PAGE of the numbered fractions is shown in the first inset (upper left); lane “a” contains molecular weight standards (bottom to top: 6.5, 14.4, 21.5, 31, 45, 66.2 kDa), and the purified protein migrates close to the 14.4 kDa standard. Lane “b” represents starting material loaded to column corresponding to guanidine·HCl-extracted inclusion bodies. Protein in the main peak (fractions 5 to 7) marked with arrow was used for protein folding after removal of guanidine·HCl by reversed-phase chromatography. The second inset (upper middle) refers to protein expression in minimal medium; lane A contains the same molecular weight standards as lane “a” in the first inset; lanes B and C correspond to insoluble protein and purified protein, respectively. Protein is labeled with 15 N and 13 .

Techniques Used: Chromatography, SDS Page, Molecular Weight, Purification, Reversed-phase Chromatography, Expressing, Labeling

19) Product Images from "Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2"

Article Title: Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2

Journal:

doi: 10.1107/S1744309107051445

Elution profile of Superose 6 gel-filtration chromatography of fourfold-crystallized Ber e 2. 8 ml fractions of the major peak (excluding the front shoulder) were collected for crystallization trials.
Figure Legend Snippet: Elution profile of Superose 6 gel-filtration chromatography of fourfold-crystallized Ber e 2. 8 ml fractions of the major peak (excluding the front shoulder) were collected for crystallization trials.

Techniques Used: Filtration, Chromatography, Crystallization Assay

20) Product Images from "Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils"

Article Title: Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils

Journal: Biomolecules

doi: 10.3390/biom7030067

Chaperone protection provided by native, fibrillar and amorphous αB-crystallin species against the ( A ) amorphous aggregation of insulin, 250 μg/mL at 37 °C in 0.1 M sodium phosphate, 20 mM DTT, pH 7.4 (0.9 chaperone: 1.0 insulin on a molar basis); and ( B ) fibrillar aggregation of RCM κ-casein 400 μg/mL, 10 μM ThT at 37 °C, pH 7.4 (0.5 chaperone: 1.0 RCM κ-casein on a molar basis). The percentage of protection provided by each chaperone is calculated from the difference between the maximal light scattering or fluorescence of the target protein alone and the target protein in the presence of the stated concentrations of αB-crystallin. Results are mean ± SE of the percentage protection given by chaperones for three experiments; p -values, derived by one-way ANOVA with Tukey post-test, are ** p
Figure Legend Snippet: Chaperone protection provided by native, fibrillar and amorphous αB-crystallin species against the ( A ) amorphous aggregation of insulin, 250 μg/mL at 37 °C in 0.1 M sodium phosphate, 20 mM DTT, pH 7.4 (0.9 chaperone: 1.0 insulin on a molar basis); and ( B ) fibrillar aggregation of RCM κ-casein 400 μg/mL, 10 μM ThT at 37 °C, pH 7.4 (0.5 chaperone: 1.0 RCM κ-casein on a molar basis). The percentage of protection provided by each chaperone is calculated from the difference between the maximal light scattering or fluorescence of the target protein alone and the target protein in the presence of the stated concentrations of αB-crystallin. Results are mean ± SE of the percentage protection given by chaperones for three experiments; p -values, derived by one-way ANOVA with Tukey post-test, are ** p

Techniques Used: Fluorescence, Derivative Assay

TEM images of αB-crystallin species formed to assess the effects of structural variation on chaperone activity: ( A ) αB Native; ( B ) αB GdnHCl Native; ( C ) αB Fibril; and ( D ) αB Amorphous. All samples were prepared as described in Table 1 . Scale bars are 200 nm. Features of importance are highlighted, including long amyloid fibrils (red arrows) and spherical aggregates of ~15 nm in diameter (yellow circles) or larger spherical aggregates (green circles).
Figure Legend Snippet: TEM images of αB-crystallin species formed to assess the effects of structural variation on chaperone activity: ( A ) αB Native; ( B ) αB GdnHCl Native; ( C ) αB Fibril; and ( D ) αB Amorphous. All samples were prepared as described in Table 1 . Scale bars are 200 nm. Features of importance are highlighted, including long amyloid fibrils (red arrows) and spherical aggregates of ~15 nm in diameter (yellow circles) or larger spherical aggregates (green circles).

Techniques Used: Transmission Electron Microscopy, Activity Assay

21) Product Images from "Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response"

Article Title: Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00754-12

Purification of conjugates. Hydrophobic interaction chromatography (phenyl-Sepharose 6 Fast Flow High Sub) of PS6B-rPspA1 conjugate: PS6B-rPspA1 produced by reductive amination (top) and PS6B-OCT-mPspA1 produced by conjugation using DMT-MM (bottom). The chromatograms of PS6B and free rPspA1 are displayed in both panels. The amounts of free PS6B and conjugated PS6B loaded in the column were 5 mg and 25 mg, respectively. Elution was with a decreasing gradient from 1 M to 0 M (NH 4 ) 2 SO 4 . The absorbance (Abs) at 490 nm and the absorbance at 280 nm correspond to the measurements for PS6B and rPspA1, respectively.
Figure Legend Snippet: Purification of conjugates. Hydrophobic interaction chromatography (phenyl-Sepharose 6 Fast Flow High Sub) of PS6B-rPspA1 conjugate: PS6B-rPspA1 produced by reductive amination (top) and PS6B-OCT-mPspA1 produced by conjugation using DMT-MM (bottom). The chromatograms of PS6B and free rPspA1 are displayed in both panels. The amounts of free PS6B and conjugated PS6B loaded in the column were 5 mg and 25 mg, respectively. Elution was with a decreasing gradient from 1 M to 0 M (NH 4 ) 2 SO 4 . The absorbance (Abs) at 490 nm and the absorbance at 280 nm correspond to the measurements for PS6B and rPspA1, respectively.

Techniques Used: Purification, Hydrophobic Interaction Chromatography, Flow Cytometry, Produced, Conjugation Assay

Opsonophagocytic assay. Pneumococcal strain 679/99 (PspA clade 3, serotype 6B, used to test the opsonic activity of anti-PS6B antibodies [Ab]) (A) and pneumococcal strain 245/00 (PspA clade 1, serotype 14, used to test the opsonic activity of anti-PspA1 antibodies) (B) were incubated with the sera from mice immunized with PS6B-rPspA1 or with PS6B-OCT-mPspA1 and a complement source. The opsonized pneumococci were incubated with peritoneal cells and plated on blood agar plates. Sera from mice immunized with saline plus Al(OH) 3 or with PS6B coadministered with rPspA1 or mPspA1 were used as controls. The numbers of CFU recovered after 20 h were compared by one-way ANOVA with Tukey's multiple-comparison test. The lines on the graph represent means. Asterisks indicate statistically significant differences (**, P
Figure Legend Snippet: Opsonophagocytic assay. Pneumococcal strain 679/99 (PspA clade 3, serotype 6B, used to test the opsonic activity of anti-PS6B antibodies [Ab]) (A) and pneumococcal strain 245/00 (PspA clade 1, serotype 14, used to test the opsonic activity of anti-PspA1 antibodies) (B) were incubated with the sera from mice immunized with PS6B-rPspA1 or with PS6B-OCT-mPspA1 and a complement source. The opsonized pneumococci were incubated with peritoneal cells and plated on blood agar plates. Sera from mice immunized with saline plus Al(OH) 3 or with PS6B coadministered with rPspA1 or mPspA1 were used as controls. The numbers of CFU recovered after 20 h were compared by one-way ANOVA with Tukey's multiple-comparison test. The lines on the graph represent means. Asterisks indicate statistically significant differences (**, P

Techniques Used: Opsonophagocytic Assay, Activity Assay, Incubation, Mouse Assay

rPspA1 secondary structure following conjugation. The protein secondary structure was assessed by CD. rPspA1 was compared to rPspA1 after modification with formaldehyde (mPspA1) and to rPspA1 after conjugation to PS6B by reductive amination (PS6B-rPspA1) or by conjugation using DMT-MM (PS6B-OCT-mPspA1). CD spectra were obtained on a Jasco J-810 spectropolarimeter at 20°C. The measurements were performed at wavelengths from 185 to 260 nm and intervals of 0.1 nm in a 0.1-cm-path cell. The secondary structure deconvolution analysis was performed with Dichroweb software, using the CDSSTR algorithm.
Figure Legend Snippet: rPspA1 secondary structure following conjugation. The protein secondary structure was assessed by CD. rPspA1 was compared to rPspA1 after modification with formaldehyde (mPspA1) and to rPspA1 after conjugation to PS6B by reductive amination (PS6B-rPspA1) or by conjugation using DMT-MM (PS6B-OCT-mPspA1). CD spectra were obtained on a Jasco J-810 spectropolarimeter at 20°C. The measurements were performed at wavelengths from 185 to 260 nm and intervals of 0.1 nm in a 0.1-cm-path cell. The secondary structure deconvolution analysis was performed with Dichroweb software, using the CDSSTR algorithm.

Techniques Used: Conjugation Assay, Modification, Software

Conjugation steps. Native PS6B (1,000 kDa) has its size reduced to 20 kDa by acid hydrolysis. Then, aldehyde groups are produced by oxidation of the PS molecule. This reactive group (aldehyde) reacts directly with amine groups on rPspA by reductive amination (the method applied in commercial vaccines) or with amine groups present in OCT. In the second case, the product, PS6B-OCT, is subsequently reacted with carboxyl groups on mPspA, intermediated by DMT-MM, to form the conjugate. In order to increase the specificity of conjugation with PS6B-OCT, PspA's lysine was previously modified with formaldehyde (mPspA).
Figure Legend Snippet: Conjugation steps. Native PS6B (1,000 kDa) has its size reduced to 20 kDa by acid hydrolysis. Then, aldehyde groups are produced by oxidation of the PS molecule. This reactive group (aldehyde) reacts directly with amine groups on rPspA by reductive amination (the method applied in commercial vaccines) or with amine groups present in OCT. In the second case, the product, PS6B-OCT, is subsequently reacted with carboxyl groups on mPspA, intermediated by DMT-MM, to form the conjugate. In order to increase the specificity of conjugation with PS6B-OCT, PspA's lysine was previously modified with formaldehyde (mPspA).

Techniques Used: Conjugation Assay, Produced, Modification

Anti-PspA immune response. (A) IgG antibody titer to rPspA1. Individual serum samples from mice ( n = 6) immunized i.p. with rPspA1 conjugated to PS6B by reductive amination (PS6B-rPspA1) or with mPspA1 conjugated to PS6B-OCT (PS6B-OCT-mPspA1) were analyzed by ELISA and compared by one-way ANOVA with Tukey's multiple-comparison test. Sera from mice immunized with the respective coadministered components or with saline plus Al(OH) 3 were used as controls. Asterisks indicate statistically significant differences (***, P
Figure Legend Snippet: Anti-PspA immune response. (A) IgG antibody titer to rPspA1. Individual serum samples from mice ( n = 6) immunized i.p. with rPspA1 conjugated to PS6B by reductive amination (PS6B-rPspA1) or with mPspA1 conjugated to PS6B-OCT (PS6B-OCT-mPspA1) were analyzed by ELISA and compared by one-way ANOVA with Tukey's multiple-comparison test. Sera from mice immunized with the respective coadministered components or with saline plus Al(OH) 3 were used as controls. Asterisks indicate statistically significant differences (***, P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

22) Product Images from "Piracetam Defines a New Binding Site for Allosteric Modulators of ?-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors §"

Article Title: Piracetam Defines a New Binding Site for Allosteric Modulators of ?-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors §

Journal: Journal of medicinal chemistry

doi: 10.1021/jm901905j

Structure of GluA3 i bound to aniracetam. (A) Dimer of GluA3 S1S2 bound to glutamate and aniracetam. One copy is shown in shades of blue and the other in shades of green. Aniracetam is shown in both orientations (one with carbons in white and the other
Figure Legend Snippet: Structure of GluA3 i bound to aniracetam. (A) Dimer of GluA3 S1S2 bound to glutamate and aniracetam. One copy is shown in shades of blue and the other in shades of green. Aniracetam is shown in both orientations (one with carbons in white and the other

Techniques Used:

23) Product Images from "Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent"

Article Title: Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent

Journal: Microbial Cell Factories

doi: 10.1186/s12934-016-0504-9

Purification of r-hGH. a FPLC chromatogram of r-hGH purification by DEAE-Sepharose ion-exchange chromatography. Blue line UV absorbance at 280 nm, brown line conductance. b FPLC chromatogram of r-hGH purification by size exclusion chromatography. c SDS-PAGE analysis of purified protein pooled after DEAE-Sepharose ion exchange chromatography. Lane 1 purified r-hGH, lane M LMW marker (97, 66, 45, 30 and 20.1 kDa). d SDS-PAGE analysis of purified protein pooled after size exclusion chromatography. Lane 1 purified r-hGH, lane M LMW marker (97, 66, 45, 30 and 20.1 kDa)
Figure Legend Snippet: Purification of r-hGH. a FPLC chromatogram of r-hGH purification by DEAE-Sepharose ion-exchange chromatography. Blue line UV absorbance at 280 nm, brown line conductance. b FPLC chromatogram of r-hGH purification by size exclusion chromatography. c SDS-PAGE analysis of purified protein pooled after DEAE-Sepharose ion exchange chromatography. Lane 1 purified r-hGH, lane M LMW marker (97, 66, 45, 30 and 20.1 kDa). d SDS-PAGE analysis of purified protein pooled after size exclusion chromatography. Lane 1 purified r-hGH, lane M LMW marker (97, 66, 45, 30 and 20.1 kDa)

Techniques Used: Purification, Fast Protein Liquid Chromatography, Ion Exchange Chromatography, Size-exclusion Chromatography, SDS Page, Marker

24) Product Images from "Probing the Allosteric Modulator Binding Site of GluR2 with Thiazide Derivatives"

Article Title: Probing the Allosteric Modulator Binding Site of GluR2 with Thiazide Derivatives

Journal: Biochemistry

doi: 10.1021/bi901127s

Binding of ALTZ (A), HFMZ (B), and IDRA-21 (C) to the GluR2 S1S2 dimer interface. The protein is colored as in . The change in bound orientation among the three compounds is shown in (D) along with a graph quantitating the movement of the rings
Figure Legend Snippet: Binding of ALTZ (A), HFMZ (B), and IDRA-21 (C) to the GluR2 S1S2 dimer interface. The protein is colored as in . The change in bound orientation among the three compounds is shown in (D) along with a graph quantitating the movement of the rings

Techniques Used: Binding Assay

Surface cavities of GluR2 S1S2. Two viewing angles of the GluR2 S1S2 dimer reveal how TCMZ (black) and HCTZ (red) bind to the interface between subunits. HCTZ's hydrobenzothiadiazide ring shifts to a deeper position in the binding pocket. The individual
Figure Legend Snippet: Surface cavities of GluR2 S1S2. Two viewing angles of the GluR2 S1S2 dimer reveal how TCMZ (black) and HCTZ (red) bind to the interface between subunits. HCTZ's hydrobenzothiadiazide ring shifts to a deeper position in the binding pocket. The individual

Techniques Used: Binding Assay

Titration of GluR2-S1S2 bound to fluorowillardiine (FW) with HFMZ. (A) Area of the bound peak for HFMZ normalized to the area of the FW bound peak (corrected for the trifluoro group of HFMZ vs . the single fluorine of FW) as a function of HFMZ concentration.
Figure Legend Snippet: Titration of GluR2-S1S2 bound to fluorowillardiine (FW) with HFMZ. (A) Area of the bound peak for HFMZ normalized to the area of the FW bound peak (corrected for the trifluoro group of HFMZ vs . the single fluorine of FW) as a function of HFMZ concentration.

Techniques Used: Titration, Concentration Assay

(A) Structure of the GluR2 S1S2 dimer in two orientations. One monomer is shown in shades of blue and the other in shades of green. ALTZ is bound in two copies to the dimer interface, and glutamate is bound to the agonist-binding site. The structures
Figure Legend Snippet: (A) Structure of the GluR2 S1S2 dimer in two orientations. One monomer is shown in shades of blue and the other in shades of green. ALTZ is bound in two copies to the dimer interface, and glutamate is bound to the agonist-binding site. The structures

Techniques Used: Binding Assay

25) Product Images from "High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension"

Article Title: High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Journal: BMC Biotechnology

doi: 10.1186/s12896-018-0485-3

Dicer expression and purification. ( a ]. ( b ) Flowchart of the Dicer expression and purification protocol. (Day 1) Cells are passaged at 0.8 × 10 6 cells/mL and incubated 24 h before transfection. (Day 2) The transfection mix is prepared and added to the cell culture, which is incubated for 72 h (37 °C, 5% CO 2 ) with shaking (100 RPM). (Day 5) Cells are harvested by centrifugation at 200×g and rinsed 3 times in cold PBS. After lysis, the cytoplasmic fraction is clarified by centrifugation, filtered and loaded on a 60-mL Q Sepharose Fast Flow column for ion-exchange chromatography. Fractions containing Dicer are pooled and loaded directly on a 5-mL HisTrap HP column for purification by immobilized metal affinity chromatography (IMAC). The Dicer-containing fractions are loaded directly on a 120-mL Superdex 200 column for purification by size-exclusion, and the fractions containing homogeneous Dicer are concentrated and stored at − 80 °C. ( c - e ) Typical chromatograms from the ( c ) ion-exchange, ( d ) affinity and ( e ) size-exclusion purifications, showing the UV absorbance at 280 nM and 260 nM along with the gradient trace. The selected fractions are highlighted by the grey area. ( f - g ) SDS-PAGE summary of the purification viewed by ( f ) Coomassie stain and ( g ) Western blot. Each lane is loaded proportionally to reflect the yield at each step ( Lane 1 : clarified lysate; Lane 2 : ion-exchange fraction pool; Lane 3 : affinity fraction pool; Lane 4 : size-exclusion fraction pool; and Lane 5 : concentrated protein). Yields were quantified from Western blot analysis, with loaded quantities of Dicer being in the linear range of detection
Figure Legend Snippet: Dicer expression and purification. ( a ]. ( b ) Flowchart of the Dicer expression and purification protocol. (Day 1) Cells are passaged at 0.8 × 10 6 cells/mL and incubated 24 h before transfection. (Day 2) The transfection mix is prepared and added to the cell culture, which is incubated for 72 h (37 °C, 5% CO 2 ) with shaking (100 RPM). (Day 5) Cells are harvested by centrifugation at 200×g and rinsed 3 times in cold PBS. After lysis, the cytoplasmic fraction is clarified by centrifugation, filtered and loaded on a 60-mL Q Sepharose Fast Flow column for ion-exchange chromatography. Fractions containing Dicer are pooled and loaded directly on a 5-mL HisTrap HP column for purification by immobilized metal affinity chromatography (IMAC). The Dicer-containing fractions are loaded directly on a 120-mL Superdex 200 column for purification by size-exclusion, and the fractions containing homogeneous Dicer are concentrated and stored at − 80 °C. ( c - e ) Typical chromatograms from the ( c ) ion-exchange, ( d ) affinity and ( e ) size-exclusion purifications, showing the UV absorbance at 280 nM and 260 nM along with the gradient trace. The selected fractions are highlighted by the grey area. ( f - g ) SDS-PAGE summary of the purification viewed by ( f ) Coomassie stain and ( g ) Western blot. Each lane is loaded proportionally to reflect the yield at each step ( Lane 1 : clarified lysate; Lane 2 : ion-exchange fraction pool; Lane 3 : affinity fraction pool; Lane 4 : size-exclusion fraction pool; and Lane 5 : concentrated protein). Yields were quantified from Western blot analysis, with loaded quantities of Dicer being in the linear range of detection

Techniques Used: Expressing, Purification, Incubation, Transfection, Cell Culture, Centrifugation, Lysis, Flow Cytometry, Ion Exchange Chromatography, Affinity Chromatography, SDS Page, Staining, Western Blot

26) Product Images from "The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex"

Article Title: The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200210028

The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1 , and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl ® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.
Figure Legend Snippet: The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1 , and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl ® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS.

Techniques Used: Purification, Translocation Assay, In Vitro, Activity Assay, Chromatography, Mass Spectrometry, Staining, SDS Page

27) Product Images from "Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1"

Article Title: Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4248-14.2015

Phosphorylation of SV2A at Cluster-2 promotes the interaction with synaptotagmin-1. Biotinylated dephosphorylated or triply phosphorylated (Ser80, Ser81, and Thr84) peptides that encompassed the Cluster-2 region of SV2A were conjugated to streptavidin–Sepharose
Figure Legend Snippet: Phosphorylation of SV2A at Cluster-2 promotes the interaction with synaptotagmin-1. Biotinylated dephosphorylated or triply phosphorylated (Ser80, Ser81, and Thr84) peptides that encompassed the Cluster-2 region of SV2A were conjugated to streptavidin–Sepharose

Techniques Used:

28) Product Images from "Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli"

Article Title: Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli

Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

doi: 10.1002/0471140864.ps0603s38

Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).
Figure Legend Snippet: Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine·HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris·Cl (pH 7.5)/4 mM guanidine·Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr). The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).

Techniques Used: Filtration, Flow Cytometry, Purification, SDS Page, Migration

Superdex 200 chromatography in guanidine·HCl of SIV gp41 27–149 . SDS-PAGE of the numbered fractions is shown in the first inset (upper left); lane “a” contains molecular weight standards (bottom to top: 6.5, 14.4, 21.5, 31, 45, 66.2 kDa), and the purified protein migrates close to the 14.4 kDa standard. Lane “b” represents starting material loaded to column corresponding to guanidine·HCl-extracted inclusion bodies. Protein in the main peak (fractions 5 to 7) marked with arrow was used for protein folding after removal of guanidine·HCl by reversed-phase chromatography. The second inset (upper middle) refers to protein expression in minimal medium; lane A contains the same molecular weight standards as lane “a” in the first inset; lanes B and C correspond to insoluble protein and purified protein, respectively. Protein is labeled with 15 N and 13 .
Figure Legend Snippet: Superdex 200 chromatography in guanidine·HCl of SIV gp41 27–149 . SDS-PAGE of the numbered fractions is shown in the first inset (upper left); lane “a” contains molecular weight standards (bottom to top: 6.5, 14.4, 21.5, 31, 45, 66.2 kDa), and the purified protein migrates close to the 14.4 kDa standard. Lane “b” represents starting material loaded to column corresponding to guanidine·HCl-extracted inclusion bodies. Protein in the main peak (fractions 5 to 7) marked with arrow was used for protein folding after removal of guanidine·HCl by reversed-phase chromatography. The second inset (upper middle) refers to protein expression in minimal medium; lane A contains the same molecular weight standards as lane “a” in the first inset; lanes B and C correspond to insoluble protein and purified protein, respectively. Protein is labeled with 15 N and 13 .

Techniques Used: Chromatography, SDS Page, Molecular Weight, Purification, Reversed-phase Chromatography, Expressing, Labeling

29) Product Images from "Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response"

Article Title: Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00754-12

Purification of conjugates. Hydrophobic interaction chromatography (phenyl-Sepharose 6 Fast Flow High Sub) of PS6B-rPspA1 conjugate: PS6B-rPspA1 produced by reductive amination (top) and PS6B-OCT-mPspA1 produced by conjugation using DMT-MM (bottom). The chromatograms of PS6B and free rPspA1 are displayed in both panels. The amounts of free PS6B and conjugated PS6B loaded in the column were 5 mg and 25 mg, respectively. Elution was with a decreasing gradient from 1 M to 0 M (NH 4 ) 2 SO 4 . The absorbance (Abs) at 490 nm and the absorbance at 280 nm correspond to the measurements for PS6B and rPspA1, respectively.
Figure Legend Snippet: Purification of conjugates. Hydrophobic interaction chromatography (phenyl-Sepharose 6 Fast Flow High Sub) of PS6B-rPspA1 conjugate: PS6B-rPspA1 produced by reductive amination (top) and PS6B-OCT-mPspA1 produced by conjugation using DMT-MM (bottom). The chromatograms of PS6B and free rPspA1 are displayed in both panels. The amounts of free PS6B and conjugated PS6B loaded in the column were 5 mg and 25 mg, respectively. Elution was with a decreasing gradient from 1 M to 0 M (NH 4 ) 2 SO 4 . The absorbance (Abs) at 490 nm and the absorbance at 280 nm correspond to the measurements for PS6B and rPspA1, respectively.

Techniques Used: Purification, Hydrophobic Interaction Chromatography, Flow Cytometry, Produced, Conjugation Assay

Opsonophagocytic assay. Pneumococcal strain 679/99 (PspA clade 3, serotype 6B, used to test the opsonic activity of anti-PS6B antibodies [Ab]) (A) and pneumococcal strain 245/00 (PspA clade 1, serotype 14, used to test the opsonic activity of anti-PspA1 antibodies) (B) were incubated with the sera from mice immunized with PS6B-rPspA1 or with PS6B-OCT-mPspA1 and a complement source. The opsonized pneumococci were incubated with peritoneal cells and plated on blood agar plates. Sera from mice immunized with saline plus Al(OH) 3 or with PS6B coadministered with rPspA1 or mPspA1 were used as controls. The numbers of CFU recovered after 20 h were compared by one-way ANOVA with Tukey's multiple-comparison test. The lines on the graph represent means. Asterisks indicate statistically significant differences (**, P
Figure Legend Snippet: Opsonophagocytic assay. Pneumococcal strain 679/99 (PspA clade 3, serotype 6B, used to test the opsonic activity of anti-PS6B antibodies [Ab]) (A) and pneumococcal strain 245/00 (PspA clade 1, serotype 14, used to test the opsonic activity of anti-PspA1 antibodies) (B) were incubated with the sera from mice immunized with PS6B-rPspA1 or with PS6B-OCT-mPspA1 and a complement source. The opsonized pneumococci were incubated with peritoneal cells and plated on blood agar plates. Sera from mice immunized with saline plus Al(OH) 3 or with PS6B coadministered with rPspA1 or mPspA1 were used as controls. The numbers of CFU recovered after 20 h were compared by one-way ANOVA with Tukey's multiple-comparison test. The lines on the graph represent means. Asterisks indicate statistically significant differences (**, P

Techniques Used: Opsonophagocytic Assay, Activity Assay, Incubation, Mouse Assay

rPspA1 secondary structure following conjugation. The protein secondary structure was assessed by CD. rPspA1 was compared to rPspA1 after modification with formaldehyde (mPspA1) and to rPspA1 after conjugation to PS6B by reductive amination (PS6B-rPspA1) or by conjugation using DMT-MM (PS6B-OCT-mPspA1). CD spectra were obtained on a Jasco J-810 spectropolarimeter at 20°C. The measurements were performed at wavelengths from 185 to 260 nm and intervals of 0.1 nm in a 0.1-cm-path cell. The secondary structure deconvolution analysis was performed with Dichroweb software, using the CDSSTR algorithm.
Figure Legend Snippet: rPspA1 secondary structure following conjugation. The protein secondary structure was assessed by CD. rPspA1 was compared to rPspA1 after modification with formaldehyde (mPspA1) and to rPspA1 after conjugation to PS6B by reductive amination (PS6B-rPspA1) or by conjugation using DMT-MM (PS6B-OCT-mPspA1). CD spectra were obtained on a Jasco J-810 spectropolarimeter at 20°C. The measurements were performed at wavelengths from 185 to 260 nm and intervals of 0.1 nm in a 0.1-cm-path cell. The secondary structure deconvolution analysis was performed with Dichroweb software, using the CDSSTR algorithm.

Techniques Used: Conjugation Assay, Modification, Software

Conjugation steps. Native PS6B (1,000 kDa) has its size reduced to 20 kDa by acid hydrolysis. Then, aldehyde groups are produced by oxidation of the PS molecule. This reactive group (aldehyde) reacts directly with amine groups on rPspA by reductive amination (the method applied in commercial vaccines) or with amine groups present in OCT. In the second case, the product, PS6B-OCT, is subsequently reacted with carboxyl groups on mPspA, intermediated by DMT-MM, to form the conjugate. In order to increase the specificity of conjugation with PS6B-OCT, PspA's lysine was previously modified with formaldehyde (mPspA).
Figure Legend Snippet: Conjugation steps. Native PS6B (1,000 kDa) has its size reduced to 20 kDa by acid hydrolysis. Then, aldehyde groups are produced by oxidation of the PS molecule. This reactive group (aldehyde) reacts directly with amine groups on rPspA by reductive amination (the method applied in commercial vaccines) or with amine groups present in OCT. In the second case, the product, PS6B-OCT, is subsequently reacted with carboxyl groups on mPspA, intermediated by DMT-MM, to form the conjugate. In order to increase the specificity of conjugation with PS6B-OCT, PspA's lysine was previously modified with formaldehyde (mPspA).

Techniques Used: Conjugation Assay, Produced, Modification

Anti-PspA immune response. (A) IgG antibody titer to rPspA1. Individual serum samples from mice ( n = 6) immunized i.p. with rPspA1 conjugated to PS6B by reductive amination (PS6B-rPspA1) or with mPspA1 conjugated to PS6B-OCT (PS6B-OCT-mPspA1) were analyzed by ELISA and compared by one-way ANOVA with Tukey's multiple-comparison test. Sera from mice immunized with the respective coadministered components or with saline plus Al(OH) 3 were used as controls. Asterisks indicate statistically significant differences (***, P
Figure Legend Snippet: Anti-PspA immune response. (A) IgG antibody titer to rPspA1. Individual serum samples from mice ( n = 6) immunized i.p. with rPspA1 conjugated to PS6B by reductive amination (PS6B-rPspA1) or with mPspA1 conjugated to PS6B-OCT (PS6B-OCT-mPspA1) were analyzed by ELISA and compared by one-way ANOVA with Tukey's multiple-comparison test. Sera from mice immunized with the respective coadministered components or with saline plus Al(OH) 3 were used as controls. Asterisks indicate statistically significant differences (***, P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

30) Product Images from "Solid-phase synthesis of molecularly imprinted polymer nanolabels: Affinity tools for cellular bioimaging of glycans"

Article Title: Solid-phase synthesis of molecularly imprinted polymer nanolabels: Affinity tools for cellular bioimaging of glycans

Journal: Scientific Reports

doi: 10.1038/s41598-019-40348-5

( A ) Equilibrium binding isotherms in water of [ 14 C]D-glucuronic acid to MIP-NP S (full circles) and NIP-NPs (empty circles) at 37 °C. Data are means of two independent experiments, with two repetitions for each point, for two batches of polymers (n = 4), error bars present standard deviations. ( B ) Relative fluorescence intensity of keratinocytes after imaging with MIP-NPs and NIP-NPs (n = 3 independent replicates for each experiment). Mean values of MIP and NIP are significantly different at 95% confidence (***p
Figure Legend Snippet: ( A ) Equilibrium binding isotherms in water of [ 14 C]D-glucuronic acid to MIP-NP S (full circles) and NIP-NPs (empty circles) at 37 °C. Data are means of two independent experiments, with two repetitions for each point, for two batches of polymers (n = 4), error bars present standard deviations. ( B ) Relative fluorescence intensity of keratinocytes after imaging with MIP-NPs and NIP-NPs (n = 3 independent replicates for each experiment). Mean values of MIP and NIP are significantly different at 95% confidence (***p

Techniques Used: Binding Assay, Fluorescence, Imaging

( A ) Size distribution as measured by dynamic light scattering in 25 mM sodium phosphate buffer pH 7.0 and ( B ) fluorescence emission spectra (λ ex = 540 nm), of MIP-NPs and NIP-NPs in water.
Figure Legend Snippet: ( A ) Size distribution as measured by dynamic light scattering in 25 mM sodium phosphate buffer pH 7.0 and ( B ) fluorescence emission spectra (λ ex = 540 nm), of MIP-NPs and NIP-NPs in water.

Techniques Used: Fluorescence

Representative epifluorescence microscope images of confluent HaCaT cells that were fixed and stained with ( A ) MIP-NPs, ( B ) FITC-labeled hyaluronic acid binding protein (HABP) and ( C ) NIP-NPs. From left to right: staining by rhodamine-labeled polymers (red) or streptavidin FITC-labeled HABP (green), cell nucleus counterstained with Hoechst (blue), the corresponding merge image and the phase contrast.
Figure Legend Snippet: Representative epifluorescence microscope images of confluent HaCaT cells that were fixed and stained with ( A ) MIP-NPs, ( B ) FITC-labeled hyaluronic acid binding protein (HABP) and ( C ) NIP-NPs. From left to right: staining by rhodamine-labeled polymers (red) or streptavidin FITC-labeled HABP (green), cell nucleus counterstained with Hoechst (blue), the corresponding merge image and the phase contrast.

Techniques Used: Microscopy, Staining, Labeling, Binding Assay

31) Product Images from "Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy"

Article Title: Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

Journal: Human Gene Therapy

doi: 10.1089/hum.2014.041

Protein profile of three serotypes of adeno-associated virus (AAV) vectors before and after AVB Sepharose affinity chromatography. (a) A typical chromatography profile, showing the separation from unbound cellular proteins of independent batches of vectors
Figure Legend Snippet: Protein profile of three serotypes of adeno-associated virus (AAV) vectors before and after AVB Sepharose affinity chromatography. (a) A typical chromatography profile, showing the separation from unbound cellular proteins of independent batches of vectors

Techniques Used: Affinity Chromatography, Chromatography

32) Product Images from "Structure of the S1S2 Glutamate Binding Domain of GluR3"

Article Title: Structure of the S1S2 Glutamate Binding Domain of GluR3

Journal: Proteins

doi: 10.1002/prot.22274

(A) Binding of [ 3 H]AMPA to the S1S2 domains of GluR2 o and GluR3 i . The K D reported a K D of 24.8 ± 1.8 nM for [ 3 H]AMPA binding to GluR2. (B) Structures of the ligands used in the binding studies, (C) The inhibition of [ 3 H]AMPA binding by agonists, partial agonists, and antagonist to the S1S2 domains of GluR2 o and GluR3 i . Except for willardiine, the IC 50 values were within 2-fold for the two subtypes: (ligand, GluR2 IC 50 /GluR3 IC 50 ; IC 50 expressed in μM) fluorowillardiine, 0.0040±.0009/0.0062±0.0014; iodowillardiine, 0.46±0.05/0.79±0.14; Cl-HIBO, 5.0±0.3/55±4; willardiine, 3.1±0.2/0.99±0.18; UBP277, 135±12/69±10. In all cases, GluR2 o is shown with filled symbols, and GluR3 i is shown with open symbols.
Figure Legend Snippet: (A) Binding of [ 3 H]AMPA to the S1S2 domains of GluR2 o and GluR3 i . The K D reported a K D of 24.8 ± 1.8 nM for [ 3 H]AMPA binding to GluR2. (B) Structures of the ligands used in the binding studies, (C) The inhibition of [ 3 H]AMPA binding by agonists, partial agonists, and antagonist to the S1S2 domains of GluR2 o and GluR3 i . Except for willardiine, the IC 50 values were within 2-fold for the two subtypes: (ligand, GluR2 IC 50 /GluR3 IC 50 ; IC 50 expressed in μM) fluorowillardiine, 0.0040±.0009/0.0062±0.0014; iodowillardiine, 0.46±0.05/0.79±0.14; Cl-HIBO, 5.0±0.3/55±4; willardiine, 3.1±0.2/0.99±0.18; UBP277, 135±12/69±10. In all cases, GluR2 o is shown with filled symbols, and GluR3 i is shown with open symbols.

Techniques Used: Binding Assay, Inhibition

33) Product Images from "A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid"

Article Title: A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid

Journal: BioMed Research International

doi: 10.1155/2015/508656

ATR FT-IR spectra of GY785 DR (a) and GY785 DRS sulfated in BMImCl medium (b).
Figure Legend Snippet: ATR FT-IR spectra of GY785 DR (a) and GY785 DRS sulfated in BMImCl medium (b).

Techniques Used:

1 H NMR spectra of GY785 DR (a) and GY785 DRS (a′) and 13 C J-modulated NMR spectra of GY785 DR (b) and GY785 DRS (b′).
Figure Legend Snippet: 1 H NMR spectra of GY785 DR (a) and GY785 DRS (a′) and 13 C J-modulated NMR spectra of GY785 DR (b) and GY785 DRS (b′).

Techniques Used: Nuclear Magnetic Resonance

Comparison of the variation of the molecular weight and the sulfur content in GY785 DRS recovered following different sulfation time.
Figure Legend Snippet: Comparison of the variation of the molecular weight and the sulfur content in GY785 DRS recovered following different sulfation time.

Techniques Used: Molecular Weight

Cumulative molecular weight fraction of GY785 DRS with the dissolution step performed under atmospheric pressure (a) or under vacuum (100 mbar) (b).
Figure Legend Snippet: Cumulative molecular weight fraction of GY785 DRS with the dissolution step performed under atmospheric pressure (a) or under vacuum (100 mbar) (b).

Techniques Used: Molecular Weight

34) Product Images from "Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils"

Article Title: Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils

Journal: Biomolecules

doi: 10.3390/biom7030067

Chaperone protection provided by native, fibrillar and amorphous αB-crystallin species against the ( A ) amorphous aggregation of insulin, 250 μg/mL at 37 °C in 0.1 M sodium phosphate, 20 mM DTT, pH 7.4 (0.9 chaperone: 1.0 insulin on a molar basis); and ( B ) fibrillar aggregation of RCM κ-casein 400 μg/mL, 10 μM ThT at 37 °C, pH 7.4 (0.5 chaperone: 1.0 RCM κ-casein on a molar basis). The percentage of protection provided by each chaperone is calculated from the difference between the maximal light scattering or fluorescence of the target protein alone and the target protein in the presence of the stated concentrations of αB-crystallin. Results are mean ± SE of the percentage protection given by chaperones for three experiments; p -values, derived by one-way ANOVA with Tukey post-test, are ** p
Figure Legend Snippet: Chaperone protection provided by native, fibrillar and amorphous αB-crystallin species against the ( A ) amorphous aggregation of insulin, 250 μg/mL at 37 °C in 0.1 M sodium phosphate, 20 mM DTT, pH 7.4 (0.9 chaperone: 1.0 insulin on a molar basis); and ( B ) fibrillar aggregation of RCM κ-casein 400 μg/mL, 10 μM ThT at 37 °C, pH 7.4 (0.5 chaperone: 1.0 RCM κ-casein on a molar basis). The percentage of protection provided by each chaperone is calculated from the difference between the maximal light scattering or fluorescence of the target protein alone and the target protein in the presence of the stated concentrations of αB-crystallin. Results are mean ± SE of the percentage protection given by chaperones for three experiments; p -values, derived by one-way ANOVA with Tukey post-test, are ** p

Techniques Used: Fluorescence, Derivative Assay

TEM images of αB-crystallin species formed to assess the effects of structural variation on chaperone activity: ( A ) αB Native; ( B ) αB GdnHCl Native; ( C ) αB Fibril; and ( D ) αB Amorphous. All samples were prepared as described in Table 1 . Scale bars are 200 nm. Features of importance are highlighted, including long amyloid fibrils (red arrows) and spherical aggregates of ~15 nm in diameter (yellow circles) or larger spherical aggregates (green circles).
Figure Legend Snippet: TEM images of αB-crystallin species formed to assess the effects of structural variation on chaperone activity: ( A ) αB Native; ( B ) αB GdnHCl Native; ( C ) αB Fibril; and ( D ) αB Amorphous. All samples were prepared as described in Table 1 . Scale bars are 200 nm. Features of importance are highlighted, including long amyloid fibrils (red arrows) and spherical aggregates of ~15 nm in diameter (yellow circles) or larger spherical aggregates (green circles).

Techniques Used: Transmission Electron Microscopy, Activity Assay

35) Product Images from "Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils"

Article Title: Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils

Journal: Biomolecules

doi: 10.3390/biom7030067

Chaperone protection provided by native, fibrillar and amorphous αB-crystallin species against the ( A ) amorphous aggregation of insulin, 250 μg/mL at 37 °C in 0.1 M sodium phosphate, 20 mM DTT, pH 7.4 (0.9 chaperone: 1.0 insulin on a molar basis); and ( B ) fibrillar aggregation of RCM κ-casein 400 μg/mL, 10 μM ThT at 37 °C, pH 7.4 (0.5 chaperone: 1.0 RCM κ-casein on a molar basis). The percentage of protection provided by each chaperone is calculated from the difference between the maximal light scattering or fluorescence of the target protein alone and the target protein in the presence of the stated concentrations of αB-crystallin. Results are mean ± SE of the percentage protection given by chaperones for three experiments; p -values, derived by one-way ANOVA with Tukey post-test, are ** p
Figure Legend Snippet: Chaperone protection provided by native, fibrillar and amorphous αB-crystallin species against the ( A ) amorphous aggregation of insulin, 250 μg/mL at 37 °C in 0.1 M sodium phosphate, 20 mM DTT, pH 7.4 (0.9 chaperone: 1.0 insulin on a molar basis); and ( B ) fibrillar aggregation of RCM κ-casein 400 μg/mL, 10 μM ThT at 37 °C, pH 7.4 (0.5 chaperone: 1.0 RCM κ-casein on a molar basis). The percentage of protection provided by each chaperone is calculated from the difference between the maximal light scattering or fluorescence of the target protein alone and the target protein in the presence of the stated concentrations of αB-crystallin. Results are mean ± SE of the percentage protection given by chaperones for three experiments; p -values, derived by one-way ANOVA with Tukey post-test, are ** p

Techniques Used: Fluorescence, Derivative Assay

TEM images of αB-crystallin species formed to assess the effects of structural variation on chaperone activity: ( A ) αB Native; ( B ) αB GdnHCl Native; ( C ) αB Fibril; and ( D ) αB Amorphous. All samples were prepared as described in Table 1 . Scale bars are 200 nm. Features of importance are highlighted, including long amyloid fibrils (red arrows) and spherical aggregates of ~15 nm in diameter (yellow circles) or larger spherical aggregates (green circles).
Figure Legend Snippet: TEM images of αB-crystallin species formed to assess the effects of structural variation on chaperone activity: ( A ) αB Native; ( B ) αB GdnHCl Native; ( C ) αB Fibril; and ( D ) αB Amorphous. All samples were prepared as described in Table 1 . Scale bars are 200 nm. Features of importance are highlighted, including long amyloid fibrils (red arrows) and spherical aggregates of ~15 nm in diameter (yellow circles) or larger spherical aggregates (green circles).

Techniques Used: Transmission Electron Microscopy, Activity Assay

36) Product Images from "Mechanisms of antagonism of the GluR2 AMPA receptor: Structure and dynamics of the complex of two willardiine antagonists with the glutamate binding domain"

Article Title: Mechanisms of antagonism of the GluR2 AMPA receptor: Structure and dynamics of the complex of two willardiine antagonists with the glutamate binding domain

Journal: Biochemistry

doi: 10.1021/bi900107m

(A) A portion of the 1 H, 15 N-HSQC (TROSY) spectrum at 10°C showing the sidechain tryptophan amides for GluR2 S1S2 bound to a variety of willardiine agonists and antagonists as well as CNQX and DNQX. The signal for W767 varies with the orientation
Figure Legend Snippet: (A) A portion of the 1 H, 15 N-HSQC (TROSY) spectrum at 10°C showing the sidechain tryptophan amides for GluR2 S1S2 bound to a variety of willardiine agonists and antagonists as well as CNQX and DNQX. The signal for W767 varies with the orientation

Techniques Used:

(A) Structures of the antagonists used in the binding study. (B) Inhibition of the binding of [ 3 H]AMPA to GluR2 S1S2 by DNQX, CNQX, UBP277, and UBP282. The IC 50 values were: 0.38 ± 0.04 μM, 0.68 ± 0.06 μM, 135 ±
Figure Legend Snippet: (A) Structures of the antagonists used in the binding study. (B) Inhibition of the binding of [ 3 H]AMPA to GluR2 S1S2 by DNQX, CNQX, UBP277, and UBP282. The IC 50 values were: 0.38 ± 0.04 μM, 0.68 ± 0.06 μM, 135 ±

Techniques Used: Binding Assay, Inhibition

The orientation of different willardiines in the GluR2 S1S2 binding site is illustrated. The residues in Lobe 1 were aligned. The positions of HW (red; 1mqj), BrW (green, 1mqh) and FW (black, 1mqi) are superimposable. However, the positions of IW (blue,
Figure Legend Snippet: The orientation of different willardiines in the GluR2 S1S2 binding site is illustrated. The residues in Lobe 1 were aligned. The positions of HW (red; 1mqj), BrW (green, 1mqh) and FW (black, 1mqi) are superimposable. However, the positions of IW (blue,

Techniques Used: Binding Assay

Structure of UBP277 bound to GluR2 S1S2
Figure Legend Snippet: Structure of UBP277 bound to GluR2 S1S2

Techniques Used:

19 F NMR spectra of antagonists bound to 5- 19 F-tryptophan-labeled GluR2 S1S2 as a function of temperature: (A) CNQX, (B) DNQX, (C) UBP277, and (D) UBP282. The signal for W767 is in chemical exchange for the CNQX-bound form but apparently not for S1S2 bound
Figure Legend Snippet: 19 F NMR spectra of antagonists bound to 5- 19 F-tryptophan-labeled GluR2 S1S2 as a function of temperature: (A) CNQX, (B) DNQX, (C) UBP277, and (D) UBP282. The signal for W767 is in chemical exchange for the CNQX-bound form but apparently not for S1S2 bound

Techniques Used: Nuclear Magnetic Resonance, Labeling

Structures of the binding sites for UBP277 (A) and UBP282 (B) on GluR2 S1S2. In each case, Lobe 1 is shown in cyan and Lobe 2 in green. Interactions between the ligand and protein sidechains are indicated. Additional interactions with the backbone are
Figure Legend Snippet: Structures of the binding sites for UBP277 (A) and UBP282 (B) on GluR2 S1S2. In each case, Lobe 1 is shown in cyan and Lobe 2 in green. Interactions between the ligand and protein sidechains are indicated. Additional interactions with the backbone are

Techniques Used: Binding Assay

37) Product Images from "High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension"

Article Title: High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Journal: BMC Biotechnology

doi: 10.1186/s12896-018-0485-3

Dicer expression and purification. ( a ) Domain architecture of Dicer. Domains were positioned using InterPro [ 56 ] and refined using available structural data [ 30 , 57 – 59 ]. ( b ) Flowchart of the Dicer expression and purification protocol. (Day 1) Cells are passaged at 0.8 × 10 6 cells/mL and incubated 24 h before transfection. (Day 2) The transfection mix is prepared and added to the cell culture, which is incubated for 72 h (37 °C, 5% CO 2 ) with shaking (100 RPM). (Day 5) Cells are harvested by centrifugation at 200×g and rinsed 3 times in cold PBS. After lysis, the cytoplasmic fraction is clarified by centrifugation, filtered and loaded on a 60-mL Q Sepharose Fast Flow column for ion-exchange chromatography. Fractions containing Dicer are pooled and loaded directly on a 5-mL HisTrap HP column for purification by immobilized metal affinity chromatography (IMAC). The Dicer-containing fractions are loaded directly on a 120-mL Superdex 200 column for purification by size-exclusion, and the fractions containing homogeneous Dicer are concentrated and stored at − 80 °C. ( c - e ) Typical chromatograms from the ( c ) ion-exchange, ( d ) affinity and ( e ) size-exclusion purifications, showing the UV absorbance at 280 nM and 260 nM along with the gradient trace. The selected fractions are highlighted by the grey area. ( f - g ) SDS-PAGE summary of the purification viewed by ( f ) Coomassie stain and ( g ) Western blot. Each lane is loaded proportionally to reflect the yield at each step ( Lane 1 : clarified lysate; Lane 2 : ion-exchange fraction pool; Lane 3 : affinity fraction pool; Lane 4 : size-exclusion fraction pool; and Lane 5 : concentrated protein). Yields were quantified from Western blot analysis, with loaded quantities of Dicer being in the linear range of detection
Figure Legend Snippet: Dicer expression and purification. ( a ) Domain architecture of Dicer. Domains were positioned using InterPro [ 56 ] and refined using available structural data [ 30 , 57 – 59 ]. ( b ) Flowchart of the Dicer expression and purification protocol. (Day 1) Cells are passaged at 0.8 × 10 6 cells/mL and incubated 24 h before transfection. (Day 2) The transfection mix is prepared and added to the cell culture, which is incubated for 72 h (37 °C, 5% CO 2 ) with shaking (100 RPM). (Day 5) Cells are harvested by centrifugation at 200×g and rinsed 3 times in cold PBS. After lysis, the cytoplasmic fraction is clarified by centrifugation, filtered and loaded on a 60-mL Q Sepharose Fast Flow column for ion-exchange chromatography. Fractions containing Dicer are pooled and loaded directly on a 5-mL HisTrap HP column for purification by immobilized metal affinity chromatography (IMAC). The Dicer-containing fractions are loaded directly on a 120-mL Superdex 200 column for purification by size-exclusion, and the fractions containing homogeneous Dicer are concentrated and stored at − 80 °C. ( c - e ) Typical chromatograms from the ( c ) ion-exchange, ( d ) affinity and ( e ) size-exclusion purifications, showing the UV absorbance at 280 nM and 260 nM along with the gradient trace. The selected fractions are highlighted by the grey area. ( f - g ) SDS-PAGE summary of the purification viewed by ( f ) Coomassie stain and ( g ) Western blot. Each lane is loaded proportionally to reflect the yield at each step ( Lane 1 : clarified lysate; Lane 2 : ion-exchange fraction pool; Lane 3 : affinity fraction pool; Lane 4 : size-exclusion fraction pool; and Lane 5 : concentrated protein). Yields were quantified from Western blot analysis, with loaded quantities of Dicer being in the linear range of detection

Techniques Used: Expressing, Purification, Incubation, Transfection, Cell Culture, Centrifugation, Lysis, Flow Cytometry, Ion Exchange Chromatography, Affinity Chromatography, SDS Page, Staining, Western Blot

38) Product Images from "Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli"

Article Title: Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

Journal: Bioprocess and Biosystems Engineering

doi: 10.1007/s00449-006-0069-9

Purification of rTfH from the PF. E. coli TG1 cells derived from a fed-batch cultivation (Run A); 37 g biomass was used for osmotic shock treatment, a Ni 2+ -affinity chromatography with Chelating Sepharose Fast Flow, b gelfiltration with Superdex 75 prep grade; pooled fraction are marked by a line
Figure Legend Snippet: Purification of rTfH from the PF. E. coli TG1 cells derived from a fed-batch cultivation (Run A); 37 g biomass was used for osmotic shock treatment, a Ni 2+ -affinity chromatography with Chelating Sepharose Fast Flow, b gelfiltration with Superdex 75 prep grade; pooled fraction are marked by a line

Techniques Used: Purification, Derivative Assay, Affinity Chromatography, Flow Cytometry

39) Product Images from "Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity"

Article Title: Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamem.2017.04.017

Panel A: Non-denaturing PAGE analysis showing a much greater mobility of apoLp-III compared to apoA-I and the chimera. Twenty μg of protein was electrophoresed in the absence of SDS on a 4–20% Tris-glycine gel. Lane 1: apoA-I, lane 2: apoLp-III cys /CT-apoA-I, lane 3: apoLp-III. Panel B: Size-exclusion chromatographic analysis. Protein (0.5 mg at a 1 mg/mL concentration) was applied to a Superdex-200 column. Elution of the proteins was monitored at 210 nm using a flow rate of 0.5 mL/min. ApoLp-III eluted as a single peak at 17 mL (dash-dotted line), while apoA-I (solid line) and the chimera (dotted line) elute much earlier at 11 mL.
Figure Legend Snippet: Panel A: Non-denaturing PAGE analysis showing a much greater mobility of apoLp-III compared to apoA-I and the chimera. Twenty μg of protein was electrophoresed in the absence of SDS on a 4–20% Tris-glycine gel. Lane 1: apoA-I, lane 2: apoLp-III cys /CT-apoA-I, lane 3: apoLp-III. Panel B: Size-exclusion chromatographic analysis. Protein (0.5 mg at a 1 mg/mL concentration) was applied to a Superdex-200 column. Elution of the proteins was monitored at 210 nm using a flow rate of 0.5 mL/min. ApoLp-III eluted as a single peak at 17 mL (dash-dotted line), while apoA-I (solid line) and the chimera (dotted line) elute much earlier at 11 mL.

Techniques Used: Polyacrylamide Gel Electrophoresis, Concentration Assay, Flow Cytometry

40) Product Images from "A diarylamine derived from anthranilic acid inhibits ZIKV replication"

Article Title: A diarylamine derived from anthranilic acid inhibits ZIKV replication

Journal: Scientific Reports

doi: 10.1038/s41598-019-54169-z

Analysis of FAM E3 intercalation into the viral dsRNA and its interaction with the activity of phage SP6 RNA polymerase. Fifteen nanomoles of dsRNA were incubated with the FAM E3 or intercalating controls (DMSO) or (DOX) for 45 minutes at room temperature. The reaction products were subjected to 1% agarose electrophoresis gel containing Ethidium Bromide followed by densitometry analysis ( a ). FAM E3 and 5 µg of purified pCCI-SP6-ZIKV amplicon was used for in vitro transcription using SP6 RNA polymerase at the presence or absence of FAM E3. Reaction products were analysed by agarose gel electrophoresis followed by densitometry analysis ( b ). Results of a representative of three independent reproducible experiments are shown.
Figure Legend Snippet: Analysis of FAM E3 intercalation into the viral dsRNA and its interaction with the activity of phage SP6 RNA polymerase. Fifteen nanomoles of dsRNA were incubated with the FAM E3 or intercalating controls (DMSO) or (DOX) for 45 minutes at room temperature. The reaction products were subjected to 1% agarose electrophoresis gel containing Ethidium Bromide followed by densitometry analysis ( a ). FAM E3 and 5 µg of purified pCCI-SP6-ZIKV amplicon was used for in vitro transcription using SP6 RNA polymerase at the presence or absence of FAM E3. Reaction products were analysed by agarose gel electrophoresis followed by densitometry analysis ( b ). Results of a representative of three independent reproducible experiments are shown.

Techniques Used: Activity Assay, Incubation, Electrophoresis, Purification, Amplification, In Vitro, Agarose Gel Electrophoresis

Related Articles

High Performance Liquid Chromatography:

Article Title: The Interface between Hepatitis B Virus Capsid Proteins Affects Self-Assembly, Pregenomic RNA Packaging, and Reverse Transcription
Article Snippet: .. Capsid and dimer fractions were separated by use of a 21-ml Superose 6 column (GE Life Sciences) mounted to a Shimadzu high-pressure liquid chromatography (HPLC) system. ..

Incubation:

Article Title: Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9
Article Snippet: .. After the incubation, the glutathione-Sepharose was washed three times with B400 buffer, washed two times with B100 buffer, and then incubated with nuclear extracts from HeLa cells at 4°C for 2 hr. .. The proteins bound to the Sepharose were eluted by lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) and subjected to Western blotting analysis using anti-Exp4 antibody.

Filtration:

Article Title: Asymmetric division triggers cell-specific gene expression through coupled capture and stabilization of a phosphatase
Article Snippet: .. Gel filtration was conducted on a 24 ml Superose 6 column (GE Healthcare, Pittsburg, PA), loading 100 µl of 1 µM SpoIIE. .. Phosphatase assays of soluble fragments of SpoIIE lacking the transmembrane domain (SpoIIE320-827 and SpoIIE320-827, V697A ) were performed using 32P phosphorylated SpoIIAA (phosphorylated by purified SpoIIAB) as a substrate.

Flow Cytometry:

Article Title: Subunit Positioning and Stator Filament Stiffness in Regulation and Power Transmission in the V1 Motor of the Manduca sexta V-ATPase
Article Snippet: .. The reconstituted sample was passed through a Superose-6 column (23 ml bed volume; GE Healthcare) linked to an Akta Purifier, developed with 20 mM Tris–HCl, 100 mM NaCl, 10 mM KCl and 2 mM 2-mercaptoethanol at pH 8.0 at a flow rate of 0.25 ml/min. ..

Purification:

Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *
Article Snippet: .. After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ). ..

Article Title: The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity
Article Snippet: .. The clarified extract was either directly loaded onto Superose 6 column following pre-clearing with 150 μl of Sepharose CL-2B (GE Healthcare, 17-0140-01) or subjected to Halo purification by incubating with 100 μl of HaloLink™ Resin (Promega, G1915) suspension overnight in 4 °C on a rotator. .. The beads were washed 3 times then subjected for protein elution with 50 units of AcTEV protease (Invitrogen, 12575-015) in PBS buffer by 8 hr incubation in 4 °C on a rotator.

Article Title: Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes
Article Snippet: .. The purification was performed using glutathione sepharose 4B according to the manufacturer’s instruction (Amersham Biosciences, Freiburg, Germany). ..

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation
Article Snippet: .. Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA). .. The purified GST-fusion proteins were desalted on Vivaspin 6 column from GE Healthcare, followed by dialysis into binding buffer (PBS, 1 mM MgCI2 , pH 7.4) to remove the free glutathione.

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  • 94
    GE Healthcare superdex 75 column
    Purification of MTC28-BAP protein. Chromatogram showing (A) Elution profile of H10-T-MTC28-BAP protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on <t>Superdex</t> 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.
    Superdex 75 Column, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superdex 75 column/product/GE Healthcare
    Average 94 stars, based on 338 article reviews
    Price from $9.99 to $1999.99
    superdex 75 column - by Bioz Stars, 2020-08
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    85
    GE Healthcare xk 26 40 columns
    Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.
    Xk 26 40 Columns, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xk 26 40 columns/product/GE Healthcare
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xk 26 40 columns - by Bioz Stars, 2020-08
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    85
    GE Healthcare empty chromatography columns xk 26 40
    Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.
    Empty Chromatography Columns Xk 26 40, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/empty chromatography columns xk 26 40/product/GE Healthcare
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Purification of MTC28-BAP protein. Chromatogram showing (A) Elution profile of H10-T-MTC28-BAP protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on Superdex 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.

    Journal: PLoS ONE

    Article Title: Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

    doi: 10.1371/journal.pone.0191315

    Figure Lengend Snippet: Purification of MTC28-BAP protein. Chromatogram showing (A) Elution profile of H10-T-MTC28-BAP protein on Ni Sepharose Fast Flow (NiFF) affinity column. Fraction numbers 6–17 were pooled (NiFF pool). (B) Elution profile of H10-T-MTC28-BAP protein on Superdex 75 gel-filtration column. Fraction numbers 16–24 were pooled (GFC pool). The GFC pool was treated with H6-TEV protease to cleave H10 tag from the protein followed by removal of cleaved tag and H6-TEV protease using Ni-affinity chromatography. (C) Elution profile of MTC28-BAP protein on Q Sepharose HP column. Fraction numbers 22–27 were pooled (QHP pool). (D) SDS-PAGE analysis of H10-T-MTC28-BAP protein at different stages during purification. The samples were analyzed by 0.1% SDS-12.5% PAGE under reducing conditions. The protein bands were visualized with Coomassie brilliant blue R-250 staining. Lane M, molecular weight marker, broad range (Bio-Rad, Hercules, CA) (shown in kDa); Lane 1, total cell after homogenization; Lane 2, High-High Speed Supernatant; Lane 3, NiFF pool; Lane 4, GFC pool; Lane 5, NiFF-TT pool (after desalting); Lane 6, QHP pool.

    Article Snippet: For H10-T-MTC28-BAP protein, gel-filtration chromatography of NiFF pool was performed on 480 ml Superdex 75 column (XK 26/100, GE Healthcare).

    Techniques: Purification, Flow Cytometry, Affinity Column, Filtration, Affinity Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight, Marker, Homogenization

    Elution profile of Superose 6 gel-filtration chromatography of fourfold-crystallized Ber e 2. 8 ml fractions of the major peak (excluding the front shoulder) were collected for crystallization trials.

    Journal:

    Article Title: Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2

    doi: 10.1107/S1744309107051445

    Figure Lengend Snippet: Elution profile of Superose 6 gel-filtration chromatography of fourfold-crystallized Ber e 2. 8 ml fractions of the major peak (excluding the front shoulder) were collected for crystallization trials.

    Article Snippet: To further purify the four-times crystallized Ber e 2, a 300 ml Superose 6 column (XK 26/70, GE Healthcare, Piscataway, NJ, USA) was used.

    Techniques: Filtration, Chromatography, Crystallization Assay

    Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.

    Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

    Article Title: Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography

    doi: 10.1016/j.jchromb.2008.02.014

    Figure Lengend Snippet: Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.

    Article Snippet: XK 26/40 columns (GE Healthcare) were packed with Q-sepharose XL resin (GE Healthcare) at room temperature using the ethanol-slurry-packing technique [ ].

    Techniques: Purification, Injection, Generated

    Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.

    Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

    Article Title: Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography

    doi: 10.1016/j.jchromb.2008.02.014

    Figure Lengend Snippet: Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.

    Article Snippet: XK 26/40 columns (GE Healthcare) were packed with Q-sepharose XL resin (GE Healthcare) at room temperature using the ethanol-slurry-packing technique [ ].

    Techniques: Purification, Injection, Generated

    Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.

    Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

    Article Title: Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography

    doi: 10.1016/j.jchromb.2008.02.014

    Figure Lengend Snippet: Chromatogram of purified hHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of hRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the hHb peak, while the “2” represents the impurity peak.

    Article Snippet: Empty chromatography columns XK 26/40 (400 mm in length, 26 mm I.D., GE Healthcare, Piscataway, NJ) were packed with the anion exchange resin Q-sepharose XL (GE Healthcare).

    Techniques: Purification, Injection, Generated

    Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.

    Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

    Article Title: Preparation of Ultrapure Bovine and Human Hemoglobin by Anion Exchange Chromatography

    doi: 10.1016/j.jchromb.2008.02.014

    Figure Lengend Snippet: Chromatogram of purified bHb Column: XK 26/40 (400 mm in length, 26 mm I.D.) was packed with 120 mL Q-sepharose XL. Injection: 50 mL of bRBC lysate was introduced into the column via Superloop. Elution: a linear gradient was generated by changing from 100% buffer A to 75% buffer B in 5 CVs (100 min). This was followed by a step gradient of 100% buffer B. The “1” in this Figure represents the bHb peak, while the “2” represents the impurity peak.

    Article Snippet: Empty chromatography columns XK 26/40 (400 mm in length, 26 mm I.D., GE Healthcare, Piscataway, NJ) were packed with the anion exchange resin Q-sepharose XL (GE Healthcare).

    Techniques: Purification, Injection, Generated