Structured Review

Bio-Rad polyvinylidene fluoride pvdf membrane
The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to <t>PVDF</t> membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. <t>Immunoblot</t> of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).
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Images

1) Product Images from "Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport"

Article Title: Plastid terminal oxidase requires translocation to the grana stacks to act as a sink for electron transport

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1719070115

The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to PVDF membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. Immunoblot of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).
Figure Legend Snippet: The effect of salt treatment on PTOX localization in thylakoid membrane of Arabidopsis and Eutrema plants subjected to 0 and 100 and 0 and 250 mM NaCl, respectively. Chloroplasts isolated 10 d after initiating salt treatment were fractionated into thylakoid membranes (T), granal thylakoid (G), stromal lamellae (L), and stroma (S). Protein samples (10 µ g) were separated by SDS/PAGE, followed by transfer to PVDF membrane, and immunoblotted with antibodies specific for PTOX ( A ). Purity of the fractions was controlled in Arabidopsis and Eutrema by separation and immunoblotting of the samples (5 μg) with antibodies specific for representative polypeptides ( B ). Coomassie brillant blue-stained SDS/PAGE gels of the thylakoid membrane fractions with chlorophyll a / b ratios given below each fraction ( C ). Linearity of the anti-PTOX immunodetection was ensured with respect to the amount of protein per lane. Immunoblot of thylakoid membranes isolated from the control plants of wild-type Eutrema presented ( D ).

Techniques Used: Isolation, SDS Page, Staining, Immunodetection

2) Product Images from "Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor"

Article Title: Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2011.01304.x

(A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human p75NTR, mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific siRNA and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.
Figure Legend Snippet: (A) Dot blots analysis of the interaction of APR-1 protein with TNFR1 human p75NTR, mouse p75NTR, FADD and rat p75NTR. A total of 5 μg of glutathione S -transferase (GST)-TNFR1 (100.0 pmol), TNFR2 (111.0 pmol), human p75NTR (106.4 pmol), mouse p75NTR (106 pmol), FADD (92.6 pmol), TrKA (113.6 pmol), TrKB (113.6 pmol), TrKC (113.6 pmol), Fas 142.8 pmol), death domain (142.8 pmol), APR-1 (100 pmol), rat p75NTR (106 pmol) or GST (192.0 pmol) were diluted in PBS and blotted onto nitrocellulose membrane and subsequently incubated overnight with in vitro transcribed and translated [ 35 S] APR-1 protein. (B) Interaction of APR-1 with P75NTR. The total cell lysates prepared from A375-APR-1 and BLM-APR-1 before and after the induction of APR-1 protein were subjected for either electrophoresis (for the detection of APR-1 and P75NTR) or for co-immunoprecipitation (IP) with either anti-P75NTR antibody or with anti-APR-1 antibody. Western blotting of IP: p75NTR for APR-1 revealed the interaction of APR-1 to P75NTR, whereas Western blotting of IP: APR-1 for P75NTR revealed the interaction of P75NTR to APR-1. β-actin was used as internal control for loading and transfer. (C) Schematic diagram of the extracellular and intracellular domains of p75NTR. Transmembrane domain, JMD and death domain. (D) GST-P75NTR recombinant proteins 1–427aa (106.4 pmol), 1–341aa (135.1 pmol), 1–311aa (147 pmol), 1–274 aa (166 pmol), 275–340aa (694.3) and 341–427aa (526.2 pmol), were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with the P75NTR domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount and the position of P75NTR recombinant proteins (left panel). (E) GST-JMD and death domain of P75NTR were separated by SDS-PAGE, and blotted on PVDF membrane and probed with in vitro transcribed and translated [ 35 S] APR-1. The interaction of APR-1 with both domains was detected by exposing the membrane to X-ray films. The coomassie-stained gel shows the amount of both JMD and death domains (left panel). (F) Western blot analysis demonstrates the expression of APR-1 by the addition of Dox to the culture medium of BLM-APR- 1, the knockdown of p75NTR by its specific siRNA and the suppression of APR-1-induced cleavage of PARP by the p75NTR siRNA. β-actin was used as internal control for loading and transfer. (G) Analysis of cell viability by counting using trypan blue staining. Rescue of APR-1-induced reduction of cell viability by the knockdown of p75NTR by siRNA for 24 or 48 hrs. Data are mean of three experiments performed separately.

Techniques Used: Incubation, In Vitro, Electrophoresis, Immunoprecipitation, Western Blot, Recombinant, SDS Page, Staining, Expressing

3) Product Images from "A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65"

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65

Journal: Biochemistry

doi: 10.1021/bi034002i

Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.
Figure Legend Snippet: Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.

Techniques Used: Labeling, Avidin-Biotin Assay, Transferring, SDS Page, Incubation

2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.
Figure Legend Snippet: 2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.

Techniques Used: SDS Page, Labeling, Two-Dimensional Gel Electrophoresis, Stripping Membranes, Electrofocusing, Silver Staining, Incubation, Avidin-Biotin Assay

Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.
Figure Legend Snippet: Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.

Techniques Used: Labeling, Avidin-Biotin Assay, SDS Page, Incubation

Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.
Figure Legend Snippet: Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.

Techniques Used: SDS Page, Staining, Labeling, Avidin-Biotin Assay, Western Blot

4) Product Images from "A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 "

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65

Journal: Biochemistry

doi: 10.1021/bi034002i

Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.
Figure Legend Snippet: Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1 , at 5 μ M, for 20 s at 4 °C; lane 4, RPE incubated with 1 , at 5 μ M, for 2 min at 4 °C; lane 5, RPE incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE incubated with 1 , at 5 μ M, for 30 min at 4 °C; lane 7, RPE incubated with 1 , at 5 μ M, for 1 h at 4 °C; and lane 8, RPE incubated with 1 , at 5 μ M, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.

Techniques Used: Labeling, Avidin-Biotin Assay, Transferring, SDS Page, Incubation

2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.
Figure Legend Snippet: 2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1 , at 10 μ M, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.

Techniques Used: SDS Page, Labeling, Two-Dimensional Gel Electrophoresis, Stripping Membranes, Electrofocusing, Silver Staining, Incubation, Avidin-Biotin Assay

Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.
Figure Legend Snippet: Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1 , at 5 μ M, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μ M, 1 h) and then 1 , at 5 μ M, for 10 min at 4 °C; lane 7, RPE labeled with 1 , at 5 μ M, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.

Techniques Used: Labeling, Avidin-Biotin Assay, SDS Page, Incubation

Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.
Figure Legend Snippet: Retinoid affinity biotinylation of RPE. (A) SDS–PAGE gradient gel (4 to 20%). Proteins were visualized by Coomassie blue staining: lane 1 , RPE labeled with 1 , 10 μ M, 1 h at 4 °C; lane 2, RPE labeled with 1 , 100 μ M, 1 h at 4 °C; and lane 3, control RPE. (B) Biotin detection of labeled proteins. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Lanes are the same as those for panel A. (C) LRAT Western blot. Proteins were transferred to a PVDF membrane, and LRAT was visualized by anti-LRAT antibody/anti-rabbit Ig-HRP/ECL. Lanes are the same as those for panel A. (D) RPE65 Western blot. Proteins were transferred to a PVDF membrane, and RPE65 was visualized by anti-RPE65 antibody/anti-rabbit Ig-HRP/ECL: lane 1, RPE labeled with 1 , 50 μ M, 1 h at 4 °C; and lane 2, control RPE.

Techniques Used: SDS Page, Staining, Labeling, Avidin-Biotin Assay, Western Blot

5) Product Images from "Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis"

Article Title: Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis

Journal: Arthritis Research & Therapy

doi: 10.1186/ar1771

Western blot analysis of anti-TIMP-2 antibodies. Lysates of THP-1 (a human monocytic cell line) and H9 (a human T-cell lymphoma) were separated in 18% Tris-glycine gel, transferred into a polyvinylidene fluoride membrane, and blotted with immunoglobulin G (IgG) fractions from a patient with rheumatoid arthritis having high levels of anti-TIMP-2 antibodies detected by ELISA. The IgG fraction visualized a band of molecular weight 22 kDa, corresponding to TIMP-2. TIMP, tissue inhibitor of metalloproteinases.
Figure Legend Snippet: Western blot analysis of anti-TIMP-2 antibodies. Lysates of THP-1 (a human monocytic cell line) and H9 (a human T-cell lymphoma) were separated in 18% Tris-glycine gel, transferred into a polyvinylidene fluoride membrane, and blotted with immunoglobulin G (IgG) fractions from a patient with rheumatoid arthritis having high levels of anti-TIMP-2 antibodies detected by ELISA. The IgG fraction visualized a band of molecular weight 22 kDa, corresponding to TIMP-2. TIMP, tissue inhibitor of metalloproteinases.

Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Molecular Weight

6) Product Images from "Analysis of Toll-Like Receptors in Human Milk: Detection of Membrane-Bound and Soluble Forms"

Article Title: Analysis of Toll-Like Receptors in Human Milk: Detection of Membrane-Bound and Soluble Forms

Journal: Journal of Immunology Research

doi: 10.1155/2019/4078671

Representative image of proteins from MFGM (a) and skimmed milk (b) fractions of colostrum (0) and/or mature milk (2) after SDS-PAGE separation. The name of bands analyzed by mass spectrometry is reported beside each lane. Bands labelled with “B” were digested from PVDF blots, while “G” bands were digested from polyacrylamide gels. S1 was digested from polyacrylamide gels, and S2, S3, S4, S5, S6, S7, S8, S9, and S10 were digested from PVDF blots.
Figure Legend Snippet: Representative image of proteins from MFGM (a) and skimmed milk (b) fractions of colostrum (0) and/or mature milk (2) after SDS-PAGE separation. The name of bands analyzed by mass spectrometry is reported beside each lane. Bands labelled with “B” were digested from PVDF blots, while “G” bands were digested from polyacrylamide gels. S1 was digested from polyacrylamide gels, and S2, S3, S4, S5, S6, S7, S8, S9, and S10 were digested from PVDF blots.

Techniques Used: SDS Page, Mass Spectrometry

7) Product Images from "Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus"

Article Title: Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus

Journal: Scientific Reports

doi: 10.1038/srep32283

Western blot of GP64 from each baculovirus. Each virus was propagated on Bm5 (BmNPVΔbgp/AcGP64/EGFP and BmNPVΔbgp/BmGP64-EGFP) or Sf-9 (BacMam 2.0) cells and partially purified. Subsequently, 1 × 10 8 or 1 × 10 7 PFU of each virus was separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to western blot analysis using rabbit anti-BmNPV GP64 polyclonal antibody. Lane 1: BmNPVΔbgp/AcGP64/EGFP, Lane 2: BmNPVΔbgp/BmGP64-EGFP, Lane 3: BacMam 2.0. Arrows indicate expressed AcGP64 or BmGP64.
Figure Legend Snippet: Western blot of GP64 from each baculovirus. Each virus was propagated on Bm5 (BmNPVΔbgp/AcGP64/EGFP and BmNPVΔbgp/BmGP64-EGFP) or Sf-9 (BacMam 2.0) cells and partially purified. Subsequently, 1 × 10 8 or 1 × 10 7 PFU of each virus was separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to western blot analysis using rabbit anti-BmNPV GP64 polyclonal antibody. Lane 1: BmNPVΔbgp/AcGP64/EGFP, Lane 2: BmNPVΔbgp/BmGP64-EGFP, Lane 3: BacMam 2.0. Arrows indicate expressed AcGP64 or BmGP64.

Techniques Used: Western Blot, Purification, SDS Page

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Nucleic Acid Electrophoresis:

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Electrophoresis:

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Article Title: Expression and Characterization of the Mycobacterium tuberculosis Serine/Threonine Protein Kinase PknB
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Staining:

Article Title: Expression and Characterization of the Mycobacterium tuberculosis Serine/Threonine Protein Kinase PknB
Article Snippet: .. PknB was separated by sodium dodecyl sulfate–7.5% polyacrylamide electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). .. The N-terminal amino acid sequence was verified after electrophoresis of samples in SDS-PAGE gels and electroblotting onto PVDF membranes.

Far Western Blot:

Article Title: Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor
Article Snippet: .. Far Western blot P75NTR protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Bio-RAD, Munche, Germany). .. The blot was placed in TEN 50 buffer (10 mM Tris-Hcl pH 8.0, 1 mM ethylenediaminetetraacetic acid [EDTA], 50 mM NaCl) and stored at 4°C overnight to allow in situ renaturation of the protein.

Western Blot:

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SDS Page:

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Article Snippet: .. PknB was separated by sodium dodecyl sulfate–7.5% polyacrylamide electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). .. The N-terminal amino acid sequence was verified after electrophoresis of samples in SDS-PAGE gels and electroblotting onto PVDF membranes.

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    p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% <t>Bis-tris</t> gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the <t>PVDF</t> membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.
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    p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% Bis-tris gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the PVDF membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.

    Journal: bioRxiv

    Article Title: Oxygen-responsive p53 tetramer-octamer switch controls cell fate

    doi: 10.1101/841668

    Figure Lengend Snippet: p53 T associates with HIF-1. (A) Anti-HIF-1α BN-PAGE immune-blot shows the rate of accumulation of different complexes of HIF-1α at 1% O 2 in HCT116p53+/+ and HCT116p53−/− cells. Purple arrows indicate HIF-1α species (M.W. 120kDa), yellow arrow shows HIF-1 complex (M.W. 212 kDa) and blue arrow suggests p53-HIF-1 complex (M.W. > HIF-1) after an extended run of lysates in 3-15% Bis-tris gradient gel. The black arrow shows higher-order HIF-1α species in HCT116p53+/+ cell line. (B) Foci like structures (yellow arrows) showing co-localization of exogenous HIF-1α (ECFP), HIF-1β (EYFP) and exogenous or endogenous p53 (DsRed Ex or TRITC) in the nucleus of the cell. Scale bar 100μm. (C) Sequestration of endogenous p53 by exogenous HIF-1 subunits in concentration-dependent manner. Scale bar 50μm. Fluorescence images are pseudo-colored and color calibration bars indicate pixel-wise fluorescence intensity. (D) Triple immune reaction-based identification of endogenous p53T-HIF-1 complex. Green arrows indicate complex with M.W. > p53-HIF-1. The black arrow identifies higher order HIF-1α species. Blue, magenta and yellow arrows indicate p53-HIF-1, p53T and HIF-1 complex respectively. Native protein standards were separated from the PVDF membrane post-transfer and stained separately by Coomassie G250. (E) Identification of endogenous p53-HIF-1 complex by cross-reaction of the same immune band against three antibodies by stepwise stripping. anti-p53 DO1 (cyan), anti-HIF-1α (green) and anti-HIF-1β (red) immune blots were merged cautiously in silico to detect cross-reactivity (white). (F) Effect of different detergent combinations on p53 or HIF-1α complexes. Blue arrows indicate p53-HIF-1 complex positions in the immune-blots. Anti-p53 immune-staining confirms dissociation of intact T from p53-HIF-1 complex by D2 detergent (magenta arrow). (G) Schematic representation of the principle of detergent displacement strategy (left panel). Anti-HIF-1α immune blot was stripped for anti-p53 immune detection and two immune blots were cautiously merged in silico to identify the dissociated p53T (magenta) and HIF-1(cyan) entities (dotted yellow circles) (right panel). Higher-order HIF-1α aggregates are shown by black arrows. For the merged anti-p53 immune-blot image, refer to Fig 6D . 3-15% Bis-Tris gradient gel was selected for proper resolution of all complexes in 1D and 2D BN-PAGE run.

    Article Snippet: The proteins were transferred to PVDF membrane (BioRad) in transfer buffer (25mM Tris, 190mM glycine and 0.1% SDS) overnight at 4°C at constant voltage (60V).

    Techniques: Polyacrylamide Gel Electrophoresis, Concentration Assay, Fluorescence, Staining, Stripping Membranes, In Silico

    Metastable p53 T operates via an oxygen-sensitive T⇀O switch. (A) Schematic representation of the CHX trap in a hypoxia gradient. (B, C) To determine metastable p53 T dynamics in response to hypoxia, CHX trap design in (A) was used to capture p53 homo-oligomerization dynamics by anti-p53 BN-PAGE immune blotting at 1, 0.1 or 5% O 2 (immune blot is shown in Fig. 5B ). To sufficiently resolve each homo-oligomer (especially T and O) 5-15% Bis-tris gradient gel (pH 7.0) was utilized. T1 represents duration for which HCT116 p53+/+ cells were exposed to hypoxia before CHX treatment. Purple arrows indicate p53 pool segregated in its constituent homo-oligomers without CHX trap. T2 represents the duration of CHX for hypoxic cells. 24h > T2 > 6h was always maintained for p53T dynamics in 0-72h T1. A red arrow in (B) shows p53 aggregating smears. Native protein standards were run in the same gel and after transfer of samples on PVDF membrane; its lane was cut and stained separately with coomassie brilliant blue G250. Due to inclusion of protein standards in 15 well gel, 60 th h sample for 1% O 2 was analyzed separately or from other replicates. SDS-PAGE based analysis of total p53 pool and GAPDH loading control of immune blots in (B, C) is shown in Fig 5A, B or Fig S3F. (D) R.A. measurements from (B, C) show oxygen-sensitive p53T via shifts in equilibrium state (5% O 2 ). Green and magenta circles correspond to on-off pattern of p53 switch deciphered at 6h. The magenta arrow shows enhanced dimerization or octamerization via T during initial durations that initiates shifts at 1 and 0.1% O 2 respectively. Values and error bars in correspond to mean and standard deviation from three independent replicates of the experiment respectively and are best represented by the immune blots in (B, C) or Fig. 5B .

    Journal: bioRxiv

    Article Title: Oxygen-responsive p53 tetramer-octamer switch controls cell fate

    doi: 10.1101/841668

    Figure Lengend Snippet: Metastable p53 T operates via an oxygen-sensitive T⇀O switch. (A) Schematic representation of the CHX trap in a hypoxia gradient. (B, C) To determine metastable p53 T dynamics in response to hypoxia, CHX trap design in (A) was used to capture p53 homo-oligomerization dynamics by anti-p53 BN-PAGE immune blotting at 1, 0.1 or 5% O 2 (immune blot is shown in Fig. 5B ). To sufficiently resolve each homo-oligomer (especially T and O) 5-15% Bis-tris gradient gel (pH 7.0) was utilized. T1 represents duration for which HCT116 p53+/+ cells were exposed to hypoxia before CHX treatment. Purple arrows indicate p53 pool segregated in its constituent homo-oligomers without CHX trap. T2 represents the duration of CHX for hypoxic cells. 24h > T2 > 6h was always maintained for p53T dynamics in 0-72h T1. A red arrow in (B) shows p53 aggregating smears. Native protein standards were run in the same gel and after transfer of samples on PVDF membrane; its lane was cut and stained separately with coomassie brilliant blue G250. Due to inclusion of protein standards in 15 well gel, 60 th h sample for 1% O 2 was analyzed separately or from other replicates. SDS-PAGE based analysis of total p53 pool and GAPDH loading control of immune blots in (B, C) is shown in Fig 5A, B or Fig S3F. (D) R.A. measurements from (B, C) show oxygen-sensitive p53T via shifts in equilibrium state (5% O 2 ). Green and magenta circles correspond to on-off pattern of p53 switch deciphered at 6h. The magenta arrow shows enhanced dimerization or octamerization via T during initial durations that initiates shifts at 1 and 0.1% O 2 respectively. Values and error bars in correspond to mean and standard deviation from three independent replicates of the experiment respectively and are best represented by the immune blots in (B, C) or Fig. 5B .

    Article Snippet: The proteins were transferred to PVDF membrane (BioRad) in transfer buffer (25mM Tris, 190mM glycine and 0.1% SDS) overnight at 4°C at constant voltage (60V).

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, SDS Page, Standard Deviation

    p53 tetramer exists as the metastable state in basal conditions. (A) Schematic representation of the homo-oligomerization trap generated by CHX (100μM) and MG132. (B) Spontaneous p53 oscillations captured by the trap in the basal state of cells. (C) Anti-p53 BN-PAGE immune blot shows p53 homo-oligomerization in basal state of U2OS cells by −CHX (only MG132 intervention) or +CHX (CHX+MG132 interventions) variants of the trap. 3-17% Bis-tris gradient gel (pH 7.0) shows p53 M, D, T, O and H.O. forms. O is observed as diffused smears. The immune density of O smear shows enhancement with an increase in MG132 dose (μM) in −CHX or +CHX variations. NativeMark protein standards were cut from the PVDF membrane after protein transfer and stained separately with coomassie brilliant blue G250 (CBB) dye. (D) R.A. calculation was performed by the densitometry of immune blots that identifies D↽T (blue arrow) and T⇀O (magenta arrow) conversion as an indicator of metastability of p53 T through −CHX and +CHX trap variants in the basal state of the cells. Immune blot shown in (C) is the best representation of the data in (D). Values and error bars in (D) represent mean and standard deviation from three independent replicates of the experiment respectively.

    Journal: bioRxiv

    Article Title: Oxygen-responsive p53 tetramer-octamer switch controls cell fate

    doi: 10.1101/841668

    Figure Lengend Snippet: p53 tetramer exists as the metastable state in basal conditions. (A) Schematic representation of the homo-oligomerization trap generated by CHX (100μM) and MG132. (B) Spontaneous p53 oscillations captured by the trap in the basal state of cells. (C) Anti-p53 BN-PAGE immune blot shows p53 homo-oligomerization in basal state of U2OS cells by −CHX (only MG132 intervention) or +CHX (CHX+MG132 interventions) variants of the trap. 3-17% Bis-tris gradient gel (pH 7.0) shows p53 M, D, T, O and H.O. forms. O is observed as diffused smears. The immune density of O smear shows enhancement with an increase in MG132 dose (μM) in −CHX or +CHX variations. NativeMark protein standards were cut from the PVDF membrane after protein transfer and stained separately with coomassie brilliant blue G250 (CBB) dye. (D) R.A. calculation was performed by the densitometry of immune blots that identifies D↽T (blue arrow) and T⇀O (magenta arrow) conversion as an indicator of metastability of p53 T through −CHX and +CHX trap variants in the basal state of the cells. Immune blot shown in (C) is the best representation of the data in (D). Values and error bars in (D) represent mean and standard deviation from three independent replicates of the experiment respectively.

    Article Snippet: The proteins were transferred to PVDF membrane (BioRad) in transfer buffer (25mM Tris, 190mM glycine and 0.1% SDS) overnight at 4°C at constant voltage (60V).

    Techniques: Generated, Polyacrylamide Gel Electrophoresis, Staining, Standard Deviation

    Lectin blot analysis of rTd neu -treated human serum (A) Normal human serum (NHS) was treated with various amounts of rTd neu at 37°C for 1 h. The resultant samples were separated on SDS-PAGE gels followed by Coomassie blue staining; (B-D) The NHS was treated with 0.2 μg rTd neu or the same amount of rTd M neu at 37°C for 3 hours. The resultant samples were separated on SDS-PAGE gels, transferred to PVDF membranes, and probed with biotin-labeled SNA ( B , 0.2 μg/ml), MAA ( C , 2 μg/ml), or ConA ( D , 0.5 μg/ml). The final concentrations of NHS in the reactions were 0.15% for SNA and ConA, and 1.5% for MAA.

    Journal: Molecular microbiology

    Article Title: A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochete Treponema denticola

    doi: 10.1111/mmi.12311

    Figure Lengend Snippet: Lectin blot analysis of rTd neu -treated human serum (A) Normal human serum (NHS) was treated with various amounts of rTd neu at 37°C for 1 h. The resultant samples were separated on SDS-PAGE gels followed by Coomassie blue staining; (B-D) The NHS was treated with 0.2 μg rTd neu or the same amount of rTd M neu at 37°C for 3 hours. The resultant samples were separated on SDS-PAGE gels, transferred to PVDF membranes, and probed with biotin-labeled SNA ( B , 0.2 μg/ml), MAA ( C , 2 μg/ml), or ConA ( D , 0.5 μg/ml). The final concentrations of NHS in the reactions were 0.15% for SNA and ConA, and 1.5% for MAA.

    Article Snippet: Equal amounts of whole cell lysates (10-50 μg) were separated on SDS-PAGE gels and then transferred to PVDF membranes (Bio-Rad).

    Techniques: SDS Page, Staining, Labeling

    Functional expression of ABCB6 variants in insect cells ( A ) Expression of the ABCB6–core domain in Sf9 insect cells. Isolated Sf9 membranes (2 μg of protein per lane) expressing β-galactosidase (β-gal, lane 1), ABCB6 (lane 2) and ABCB6–core (lane 3) were separated by SDS/PAGE (7.5% gel) and were electroblotted on to PVDF membranes. Immunoblotting was performed using monoclonal anti-ABCB6-567 antibody as described in the Experimental section. Membrane proteins are only core–glycosylated in insect cells [ 6 ], which is consistent with the apparent molecular mass of 95 kDa, corresponding to under-glycosylated ABCB6. ( B ) TMD 0 is not required for ATP binding. Isolated Sf9 membranes expressing β-galactosidase (lane 1), ABCB6 (lane 2) and ABCB6–core (lane 3) were incubated with 5 μM 8-azido-[α- 32 P]ATP under non-hydrolytic conditions (at 4°C) for 5 min, followed by UV irradiation in the presence of the labelled nucleotide as described in the Experimental section. ( C ) TMD 0 is not required for ATP hydrolysis. Isolated Sf9 membranes expressing β-galactosidase (lane 1), ABCB6 (lane 2) and ABCB6–core (lane 3) were incubated with 5 μM 8-azido-[α- 32 P]ATP and 0.4 mM sodium orthovanadate under catalytic conditions (at 37°C) as described in the Experimental section. Both the full-length and the N-terminally truncated ABCB6–core are capable of ATP binding and hydrolysis. The lower-molecular-mass bands seen in lane 2 correspond to proteolytic fragments and products of vanadate-induced photocleavage [ 60 , 61 ]. ( D ) Mutation of the conserved Walker A lysine is compatible with ATP binding but abolishes nucleotide trapping of ABCB6. Isolated Sf9 membranes expressing-ABCB6-K 629 M were incubated with 5–50 μM 8-azido-[α- 32 P]ATP under non-hydrolytic (left) and hydrolytic (right) conditions as described in the Experimental section.

    Journal: Biochemical Journal

    Article Title: Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

    doi: 10.1042/BJ20141085

    Figure Lengend Snippet: Functional expression of ABCB6 variants in insect cells ( A ) Expression of the ABCB6–core domain in Sf9 insect cells. Isolated Sf9 membranes (2 μg of protein per lane) expressing β-galactosidase (β-gal, lane 1), ABCB6 (lane 2) and ABCB6–core (lane 3) were separated by SDS/PAGE (7.5% gel) and were electroblotted on to PVDF membranes. Immunoblotting was performed using monoclonal anti-ABCB6-567 antibody as described in the Experimental section. Membrane proteins are only core–glycosylated in insect cells [ 6 ], which is consistent with the apparent molecular mass of 95 kDa, corresponding to under-glycosylated ABCB6. ( B ) TMD 0 is not required for ATP binding. Isolated Sf9 membranes expressing β-galactosidase (lane 1), ABCB6 (lane 2) and ABCB6–core (lane 3) were incubated with 5 μM 8-azido-[α- 32 P]ATP under non-hydrolytic conditions (at 4°C) for 5 min, followed by UV irradiation in the presence of the labelled nucleotide as described in the Experimental section. ( C ) TMD 0 is not required for ATP hydrolysis. Isolated Sf9 membranes expressing β-galactosidase (lane 1), ABCB6 (lane 2) and ABCB6–core (lane 3) were incubated with 5 μM 8-azido-[α- 32 P]ATP and 0.4 mM sodium orthovanadate under catalytic conditions (at 37°C) as described in the Experimental section. Both the full-length and the N-terminally truncated ABCB6–core are capable of ATP binding and hydrolysis. The lower-molecular-mass bands seen in lane 2 correspond to proteolytic fragments and products of vanadate-induced photocleavage [ 60 , 61 ]. ( D ) Mutation of the conserved Walker A lysine is compatible with ATP binding but abolishes nucleotide trapping of ABCB6. Isolated Sf9 membranes expressing-ABCB6-K 629 M were incubated with 5–50 μM 8-azido-[α- 32 P]ATP under non-hydrolytic (left) and hydrolytic (right) conditions as described in the Experimental section.

    Article Snippet: Isolated Sf9 membranes were run on 7.5% Laemmli-type SDS gels and the proteins were electroblotted on to PVDF membranes (Bio-Rad).

    Techniques: Functional Assay, Expressing, Isolation, SDS Page, Binding Assay, Incubation, Irradiation, Mutagenesis

    Mobility of G to L Mutants of CEACAM1-4S Resolved by BN-PAGE. Protein lysates were prepared and resolved by BN-PAGE as described under materials and methods . Proteins were transferred onto PVDF membranes and probed with monoclonal antibody 9.2. When separated on native gels, wild type CEACAM1-4S and the single G mutants migrated with an apparent molecular mass that was approximately 100 kDa higher than the double glycine mutant.

    Journal: PLoS ONE

    Article Title: The Transmembrane Domain of CEACAM1-4S Is a Determinant of Anchorage Independent Growth and Tumorigenicity

    doi: 10.1371/journal.pone.0029606

    Figure Lengend Snippet: Mobility of G to L Mutants of CEACAM1-4S Resolved by BN-PAGE. Protein lysates were prepared and resolved by BN-PAGE as described under materials and methods . Proteins were transferred onto PVDF membranes and probed with monoclonal antibody 9.2. When separated on native gels, wild type CEACAM1-4S and the single G mutants migrated with an apparent molecular mass that was approximately 100 kDa higher than the double glycine mutant.

    Article Snippet: After running at 150 V for approximately 30 min, the cathode buffer was changed from 0.02% to 0.002% G-250 and electrophoresis at 150 V was continued for an additional 120 min. BN-PAGE gels were immunoblotted onto PVDF membranes (Bio-Rad).

    Techniques: Polyacrylamide Gel Electrophoresis, Mutagenesis