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Bio-Rad polyvinylidene difluoride pvdf
<t>Immunoblot</t> analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a <t>PVDF</t> membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.
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1) Product Images from "Demonstration of the Genetic Stability and Temporal Expression of Select Members of the Lyme Disease Spirochete OspF Protein Family during Infection in Mice"

Article Title: Demonstration of the Genetic Stability and Temporal Expression of Select Members of the Lyme Disease Spirochete OspF Protein Family during Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.69.8.4831-4838.2001

Immunoblot analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a PVDF membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.
Figure Legend Snippet: Immunoblot analysis of r-OspF protein family members expressed in E. coli . Each member of the ospF gene family was cloned and expressed as an S-tag fusion protein using ligase-independent cloning methods as described in the text. Proteins from E. coli cultures that were induced to express the r-proteins with IPTG were fractionated by SDS-PAGE and transferred to a PVDF membrane by electroblotting. The membrane on the left was screened with anti-S-Tag protein HRP conjugate, while the membrane on the right was screened with a polyclonal anti-OspF antiserum (generated with a gene of B. burgdorferi N40 origin). Immunoblot methods are described in the text. Molecular size standards are indicated on the left.

Techniques Used: Clone Assay, Ligase Independent Cloning, SDS Page, Generated

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Nucleic Acid Electrophoresis:

Article Title: Proteomic Analysis of Lymphoblastoid Cells Derived from Monozygotic Twins Discordant for Bipolar Disorder: A Preliminary Study
Article Snippet: .. Equal concentration (5 μg per lane) of proteins from control and case samples were separated by 12% or 4–15% SDS-polyacrylamide gel electrophoresis and transferred onto Immun-Blot PVDF membranes (Bio-Rad) using a mini Trans-blot Cell (Bio-Rad). .. After transfer, the blotted membrane were blocked with 4% w/v ECL Advance Blocking Reagent (GE Healthcare Bio-Sciences) in phosphate-buffered saline containing 0.1% Tween20 (PBST) (MP Biomedicals Inc., Santa Ana, CA) at 4°C overnight and incubated with primary antibody in PBST with 4% w/v skim milk for 1 h at room temperature.

Purification:

Article Title: Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins
Article Snippet: .. Purified C. difficile toxins A and B (80 µg each) were separated on 6% polyacrylamide electrophoresis (PAGE) gels and transferred onto Immun-Blot PVDF membrane (BioRad, Hercules, CA) using a Trans-Blot cell (BioRad) transfer apparatus. ..

Electrophoresis:

Article Title: Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins
Article Snippet: .. Purified C. difficile toxins A and B (80 µg each) were separated on 6% polyacrylamide electrophoresis (PAGE) gels and transferred onto Immun-Blot PVDF membrane (BioRad, Hercules, CA) using a Trans-Blot cell (BioRad) transfer apparatus. ..

Concentration Assay:

Article Title: Proteomic Analysis of Lymphoblastoid Cells Derived from Monozygotic Twins Discordant for Bipolar Disorder: A Preliminary Study
Article Snippet: .. Equal concentration (5 μg per lane) of proteins from control and case samples were separated by 12% or 4–15% SDS-polyacrylamide gel electrophoresis and transferred onto Immun-Blot PVDF membranes (Bio-Rad) using a mini Trans-blot Cell (Bio-Rad). .. After transfer, the blotted membrane were blocked with 4% w/v ECL Advance Blocking Reagent (GE Healthcare Bio-Sciences) in phosphate-buffered saline containing 0.1% Tween20 (PBST) (MP Biomedicals Inc., Santa Ana, CA) at 4°C overnight and incubated with primary antibody in PBST with 4% w/v skim milk for 1 h at room temperature.

Incubation:

Article Title: A role for the dehydrogenase DHRS7 (SDR34C1) in prostate cancer
Article Snippet: .. Lysates were separated by a 12.5% Tris-glycine SDS-polyacrylamide gel, and transferred to Immun-Blot® polyvinylidene difluoride membranes (162-0177; Bio-Rad Laboratories, Hercules, CA) at constant 230 mA for 1 h. For detection of DHRS7, the membrane was blocked using 2% milk (v/v) for 1 h at room temperature, followed by incubation with the mouse anti-human DHRS7 polyclonal antibody (ab69348; Abcam, Cambridge, UK) at a dilution of 1:500 (v/v) in 2% milk (v/v), overnight at 4°C. .. After washing with Tris-buffered saline (20 mmol/L Tris-base, 140 mmol/L NaCl) containing 0.1% Tween-20 (v/v) (TBS-T), the membrane was subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Immuno Research, Suffolk, UK) for 1 h at room temperature.

Polyacrylamide Gel Electrophoresis:

Article Title: Denatured G-Protein Coupled Receptors as Immunogens to Generate Highly Specific Antibodies
Article Snippet: .. Western blot analysis Protein lysates prepared from Pichia pastoris cell extracts, GPCR-expressing CHO membranes, mouse olfactory bulb and cerebellum cell membranes or human spermatozoa were run on sodium dodecyl sulfate (SDS) 10% polyacrylamide gel electrophoresis and transferred onto a PVDF immun-blot membrane (Bio-Rad Laboratories, Hercules, CA). .. Receptors were probed with GPCR-primed mouse immune sera or when indicated with anti-c-myc monoclonal antibody (clone 9E10, diluted at 1∶1000, Sigma).

Article Title: Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins
Article Snippet: .. Purified C. difficile toxins A and B (80 µg each) were separated on 6% polyacrylamide electrophoresis (PAGE) gels and transferred onto Immun-Blot PVDF membrane (BioRad, Hercules, CA) using a Trans-Blot cell (BioRad) transfer apparatus. ..

Western Blot:

Article Title: Denatured G-Protein Coupled Receptors as Immunogens to Generate Highly Specific Antibodies
Article Snippet: .. Western blot analysis Protein lysates prepared from Pichia pastoris cell extracts, GPCR-expressing CHO membranes, mouse olfactory bulb and cerebellum cell membranes or human spermatozoa were run on sodium dodecyl sulfate (SDS) 10% polyacrylamide gel electrophoresis and transferred onto a PVDF immun-blot membrane (Bio-Rad Laboratories, Hercules, CA). .. Receptors were probed with GPCR-primed mouse immune sera or when indicated with anti-c-myc monoclonal antibody (clone 9E10, diluted at 1∶1000, Sigma).

SDS Page:

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Article Title: Antibody Responses against Xenotropic Murine Leukemia Virus-Related Virus Envelope in a Murine Model
Article Snippet: .. Samples were separated by 12% SDS-PAGE, transferred to Immun-Blot™ PVDF membrane (Bio-Rad, Hercules, CA), blocked with 5% BSA in TBS-T buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.4) and probed with R187 anti-Gag monoclonal antibody or 83A25 anti-Env monoclonal antibody . ..

Molecular Weight:

Article Title: Translating the message: Karlodinium veneficum possesses an expanded toolkit of protein translation initiation factors
Article Snippet: .. The samples were separated by 17.5 % high-Tris SDS-PAGE using PAGEruler pre-stained molecular weight ladder (Fermentas) as a guide and transferred to Immun-blot PVDF (BioRad) using a Criterion blotter (BioRad) for 30 min at 100 V in 20 % methanol Towbin buffer (BioRad). .. Labeled proteins were visualized on the PVDF membrane using a Storage Phosphor screen (Molecular Dynamics) and imaged with a Typhoon 9410 Variable Mode Imager (GE Healthcare).

Article Title: BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy
Article Snippet: .. Twenty-five μg of proteins were separated according to molecular weight on NuPAGE Novex Bis-Tris 4–12% precast gels (Invitrogen, NW04120BOX and NP0321BOX) and electrotransferred to ImmobilonTM PVDF membranes (Bio-Rad, 162-0176 and 162-0177). .. Nonspecific binding sites were blocked with 5% nonfat powdered milk (w:v) plus 0.05% Tween 20 (v:v; Sigma, P2287 or P1379) in Tris-Buffered Saline (50 mM Tris-Cl, pH 7.6; 150 mM NaCl) for 1 h, followed by overnight incubation at 4°C with primary antibodies.

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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