C. jejuni cells were disrupted by bead beating (Bead Ruptor 12, Omni International) at maximal intensity for 30 s and the crude cell extracts were used for SDS/PAGE. For Western blot analysis, proteins were electroblotted on to nitrocellulose membranes (Amersham Protran 0.45 μm NC, GE Healthcare) for 35 min at 15 V using the Transblot SD semi-dry transfer apparatus (Bio-Rad Laboratories). His-tagged protein was detected with His-tag monoclonal antibody (#70796-3, Novagen) and Goat Anti-Mouse IgG horseradish peroxidase (HRP) Conjugate (#71045-3, Novagen) via the associated peroxidase activity using either chloronaphthol or the SignalFire ECL Reagent kit (Cell Signaling Technology).
Amersham protran 0.45 μm nc
Manufactured by GE Healthcare
Sourced in Germany
The Amersham Protran 0.45 μm NC is a nitrocellulose membrane used for protein transfer and immobilization in western blotting applications. It has a pore size of 0.45 micrometers and is suitable for the transfer of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Blue dextran, by Merck Group (2 mentions)
Aldolase, by Merck Group (2 mentions)
Carbonic anhydrase, by Merck Group (2 mentions)
Conalbumin, by Merck Group (2 mentions)
Kta purifier, by Cytiva (2 mentions)
Penta his antibody horseradish peroxidase hrp conjugate, by Qiagen (2 mentions)
Hiload 16 600 superdex 75 pg, by GE Healthcare (2 mentions)
Rnase a, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Trans blot sd semidry transfer apparatus, by Bio-Rad (1 mentions)
Bead ruptor 12, by Omni International (1 mentions)
Tank blotter, by Bio-Rad (1 mentions)
Startingblock t20 tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Mouse anti β tubulin, by Merck Group (1 mentions)
Mirvana paris kit, by Thermo Fisher Scientific (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Anti phospho nf κb p65 ser536 93h1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Image studio lite software, by LI COR (1 mentions)
11 protocols using amersham protran 0.45 μm nc
1
Western Blot Analysis of His-Tagged Proteins
2
Protein Gel Electrophoresis and Immunoblotting
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Samples were run on gels consisting of a 4% w/v acrylamide stacking gel [4% w/v acrylamide, 0.125 M Tris-HCl pH 6.8, 0.2% v/v Tetramethylethylenediamine (TEMED) and 0.08% w/v ammonium persulphate (APS)] and 10% w/v acrylamide separating gel [10% w/v acrylamide, 0.375 M Bis-Tris pH 6.8, 1% v/v tetramethylethylenediamine (TEMED) and 0.05% w/v ammonium persulphate (APS)] in MOPS buffer (50 mM MOPS, 50 mM Tris, 1 mM EDTA, 0.1% w/v SDS) at 90-120 V. For Coomassie staining, gels were stained with InstantBlue™ Ultrafast Protein Stain (ISB1L, Sigma-Aldrich) according to the manufacturer's instructions and the gels were imaged using LICOR Odyssey CLx. For immunoblot analysis, proteins were electrophoretically transferred onto nitrocellulose membranes (Amersham Protran 0.45 μm NC; GE Healthcare) at 90 V for 90 min on ice in transfer buffer [48 mM Tris/HCl, 39 mM glycine, 20% v/v methanol]. Transferred membranes were blocked with 5% w/v non-fat dry milk dissolved in TBS-T [20 mM Tris/HCl, pH 7.5, 150 mM NaCl and 0.1% v/v Tween 20] at room temperature for 1 h. Membranes were then incubated with primary antibodies overnight at 4°C. After washing membranes in TBS-T 3x15 min, membranes were incubated with secondary antibodies at room temperature for 1 h. After washing membranes in TBS-T 3x15 min membranes were scanned using LICOR Odyssey CLx.
3
Protein Extraction and Western Blot Analysis
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Cells were harvested and immediately homogenized using mirVana Paris Kit (Applied Biosystems) following the manufacturer’s protocol. Protein content was determined using BCA Protein Assay Kit (Novagen). 30 μg of protein was loaded on a 10% SDS-PAGE gel followed by transfer to nitrocellulose (Amersham Protran 0.45μm NC; GE Healthcare Life Science) in a Tank Blotter (Biorad). Blots were blocked with StartingBlock T20 (TBS) Blocking Buffer (Thermo Fisher Scientific) for 1 h. Filters with specific antibody solutions were incubated overnight in blocking solution (Rabbit anti TNS4 1:500; Abcam), followed by 2x TBST and 3x TBS washing steps and incubation with biotinylated antibody solutions for 1 h. After TNS4 visualisation the membrane was stripped using Restore Western Blot Stripping buffer (Thermo Fisher Scientific) for 30 min, blocked and incubated overnight in mouse anti β-Tubulin (1:5000; Sigma) followed by the procedure described above. For visualization, blots were washed again and developed by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
4
Cytoplasmic and Nuclear Protein Extraction
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For cytoplasmic and nuclear extracts, Caco‐2 cells were harvested, washed in cold PBS twice, and centrifuged at 3,300 g for 5 min in cold room to collect pellets. Pellets were resuspended in double cell volume of cytoplasmic extract (CE) buffer [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.3% NP‐40, and 1× protease inhibitor cocktail (Thermo Scientific™)], incubated on ice for 10 min and centrifuged at 800 g for 5 min to obtain the supernatants as cytoplasmic fraction. The pellets (containing the nuclei) were resuspended in equal volume of nuclear extract (NE) buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 25% glycerol, and 1× protease inhibitor cocktail), incubated on ice for 10 min, and centrifuged at 16,100 g for 5 min to obtain the supernatants as nuclear fraction. The protein concentrations were measured by Bradford protein assay (Protein Reagent, Bio‐Rad). For Western blots, cytoplasmic and nuclear proteins were fractionated on 10% SDS–PAGE and transferred to nitrocellulose blotting membranes (Amersham™Protran™ 0.45 μm NC, GE Healthcare, Life science). Membranes were incubated with anti‐phospho‐NF‐κB p65 (Ser536; (93H1), Cell Signaling, #3033), 1:1,000, Lamin B1‐Nuclear Envelope Marker (Abcam, ab16048) 1:5,000, and anti‐β‐actin (Cell Signaling, #4970) 1:1,000.
5
VDAC1 Protein Expression Analysis by Western Blot
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1 x 106 cells for each line were collected and lysed in extraction buffer (50 mM Tris, 150 mM NaCl, EDTA 1 mM, 1% TRITON X-100, pH 7.4, and protease inhibitors). After lysis, the supernatant was collected, and the protein content was estimated with the Bradford reagent with bovine serum albumin (BSA) as a standard. 50 μg of total protein of each sample were separated using SDS/PAGE electrophoresis, transferred to a nitrocellulose membrane (AmershamProtran 0.45 μM NC; GE Healthcare Life Sciences), and blocked in 5 % (w/v) BSA at room temperature for 1h. The membranes were incubated overnight at 4°C with primary antibodies against VDAC1 and β-Actin (1:5000, #2146; Cell Signaling Technology) after the washing step with secondary antibodies HRP-conjugate Goat anti-Mouse IgG (H+L) (1:5000, #89842, Invitrogen), HRP-conjugate Goat anti-Rabbit IgG (H+L) (1:5000, #31460, Invitrogen). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate. The membranes were read using the Azure c600 Imaging System (Azure Biosystems). For the western-blot analysis, data were analyzed with Image-StudioLite software (Li-Cor Biosciences), and β-Actin was used as an internal loading control. Data were statistically analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test.
6
Analysis of Purified Proteins
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The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His10-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
7
Western Blot Analysis of ABCG2 Protein
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Proteins were separated by SDS-PAGE (4–15% Criterion TGX Precast Gel, Bio-Rad), and transferred to a nitrocellulose membrane (Amersham Protran 0.45 μm NC, GE Healthcare). Immunoblot analysis was performed using primary antibody against ABCG2 (BXP-53, Enzo Life Sciences Inc., 1:500) and was subjected to further incubation with HRP-conjugated secondary antibody raised against rat IgG (Jackson ImmunoResearch Laboratories Inc., 1:10,000) as previously described11 (link). Chemiluminescence signal was detected using LI-COR Odyssey Fc Imaging system (Image Studio 5.2). The bands were analyzed using LI-COR Image Studio Lite Ver 5.2 software for quantification of western blot signals. As a membrane marker, immunoblot analysis was performed using primary antibody against ATP1A1 (sodium potassium ATPase alpha 1, Novus Biologicals, Littleton, CO, 1:5000). As the secondary antibody, HRP-conjugated secondary antibody raised against mouse IgG (Jackson ImmunoResearch Laboratories Inc., 1:10,000) was used.
8
Protein Quantification by Western Blot
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Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Protran 0.45 μm NC, GE Healthcare, Pittsburgh, PA, USA). Membranes were blocked using 3% bovine albumin in TBST (10 mM Tris-Cl pH 7.5, 100 mM NaCl plus 0.1% Tween-20) and incubated with primary antibodies overnight at 4°C. Incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies was done at room temperature for 1 h, followed by washing with TBST. Chemiluminescence signals were detected using LAS-300 Imaging system (FujiFilm, Düsseldorf, Germany) and densitometrically evaluated with the help of Aida Image Analyzer 3.52 software (Raytest, Straubenhardt, Germany).
9
Analysis of Purified Proteins
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The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His10-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min−1 with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M−1 cm−1 (StyI) and 48,150 M−1 cm−1 (StyJ) as well as molecular weights of 29,836.3 g mol−1 (StyI) and 30,095.6 g mol−1 (StyJ) as predicted by Expasy ProtParam (56 (link)).
10
Immunoblotting Protein Detection Protocol
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All cell treatments
were carried out as described in figure legends, to a final DMSO concentration
of 0.1% (v/v). Lysates were quantified. Immunoblotting was performed
using standard procedures, described in brief below. Lysate concentration
was quantified by Bradford Assay, and 10 μg of lysate was loaded
in the LDS sample buffer for SDS-PAGE electrophoresis on Novex 4–12%
Bis–Tris gels. Proteins were electrophoretically transferred
onto nitrocellulose membranes (Amersham Protran 0.45 μm NC;
GE Healthcare) at 80 V for 80 min on ice in transfer buffer. Transferred
membranes were blocked with 5% (w/v) nonfat dry milk dissolved in
TBS-T [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20]
at RT for 30 min, before incubation with the primary antibody overnight
at 4 °C. The signal was produced with near-infrared secondary
antibodies and detected using a Licor Biosciences Odyssey System,
and the signal was quantified in Image Studio Lite.
were carried out as described in figure legends, to a final DMSO concentration
of 0.1% (v/v). Lysates were quantified. Immunoblotting was performed
using standard procedures, described in brief below. Lysate concentration
was quantified by Bradford Assay, and 10 μg of lysate was loaded
in the LDS sample buffer for SDS-PAGE electrophoresis on Novex 4–12%
Bis–Tris gels. Proteins were electrophoretically transferred
onto nitrocellulose membranes (Amersham Protran 0.45 μm NC;
GE Healthcare) at 80 V for 80 min on ice in transfer buffer. Transferred
membranes were blocked with 5% (w/v) nonfat dry milk dissolved in
TBS-T [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20]
at RT for 30 min, before incubation with the primary antibody overnight
at 4 °C. The signal was produced with near-infrared secondary
antibodies and detected using a Licor Biosciences Odyssey System,
and the signal was quantified in Image Studio Lite.
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