SDS‐PAGE and subsequent western blotting analyses were performed as previously described.29 Cell lysates were prepared by solubilizing cells in radio‐immunoprecipitation assay (RIPA) buffer (50 mM Tris‐HCl [pH 7.5] containing 150 mM NaCl, 1% NP‐40, and 0.1% sodium deoxycholate) supplemented with protease‐inhibitor and phosphatase‐inhibitor cocktails (Sigma). Samples were then mixed at a 1:4 ration with 5 × sample buffer (300 mM Tris‐HCl [pH 6.8] containing 10% 2‐mercaptoethanol, 10% SDS, 50% glycerol and 0.05% Coomassie Brilliant Blue) and boiled for 5 min. The resulting samples (20 μg protein/lane) were subsequently separated by SDS‐PAGE using pre‐cast 5%–20% Tris‐glycine gradient gels (Atto Corporation, Tokyo, Japan) and transferred to PVDF membranes (Wako Pure Chemical). Membranes were blocked by incubation with TBS containing 0.1% Tween 20 (TBS‐T) and 5% skim milk (Megmilk Snow Bland, Tokyo, Japan) or BSA (Sigma) for 1 h, probed with the appropriate primary antibodies diluted with Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan) at 4°C, washed extensively with TBS‐T, and incubated with HRP‐conjugated secondary antibodies (1:5000 dilution) (GE Healthcare, Little Chalfont, UK). Finally, the membranes were washed, developed by incubation with ECL detection reagents (GE Healthcare) and exposed to Hyperfilm ECL (GE Healthcare).
Pvdf membrane
Manufactured by Fujifilm
Sourced in Japan
PVDF membrane is a type of microporous polymer membrane made from polyvinylidene fluoride. It is a durable and chemically resistant material commonly used in laboratory filtration and separation applications.
Automatically generated - may contain errors
Lab products found in correlation
Blocking one, by Nacalai Tesque (5 mentions)
X ray film, by Fujifilm (3 mentions)
Canoscan lide400 image scanner, by Canon (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sodium dodecyl sulfate polyacrylamide gels, by Nacalai Tesque (2 mentions)
Un scan it software, by Silk Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Can get signal immunoreaction enhancer solution, by Toyobo (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
β actin, by Proteintech (1 mentions)
Denaturing sample buffer, by Nacalai Tesque (1 mentions)
Sample buffer, by Nacalai Tesque (1 mentions)
Nuclear cytosolic fractionation kit, by Cell Biolabs (1 mentions)
Rx u fuji x ray film, by Fujifilm (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
17 protocols using pvdf membrane
1
SDS-PAGE and Western Blotting Analysis
2
Curcumin's Effect on Membrane Protein Binding
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A membrane-binding assay was conducted to determine whether curcumin had an effect on protein binding to target membranes. Sheep erythrocytes were lyzed in 20 mM MgCl2, broken by sonication, and then, centrifuged at 3,000 × g for 10 min, and the supernatants were centrifuged at 20,000 × g for 2 h. Precipitate was added to the mixture containing pre-incubated LLO or mutants with various concentrations of curcumin for 30 min, following incubation for a further 15 min.
A total of 10 µl of each sample was loaded onto a 12% SDS-PAGE after boiling in Laemmli sample buffer. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Wako Pure Chemical Industries Ltd., Osaka, Japan) and blocked with 5% non-fat dried milk at room temperature for 2 h. To test the LLO expression, a rabbit antibody reactive to LLO (Abcam) was diluted at 1:2,000 and applied for 2 h for use as the primary antibody, and a horseradish peroxidase-conjugated antibody (Proteintech) at 1:3,000 was applied for another 2 h as the secondary antibody. Beta-actin (Proteintech) was used as an internal control in this assay, and it was used according to the recommended dilution.
The blots were detected using Amersham ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK).
A total of 10 µl of each sample was loaded onto a 12% SDS-PAGE after boiling in Laemmli sample buffer. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Wako Pure Chemical Industries Ltd., Osaka, Japan) and blocked with 5% non-fat dried milk at room temperature for 2 h. To test the LLO expression, a rabbit antibody reactive to LLO (Abcam) was diluted at 1:2,000 and applied for 2 h for use as the primary antibody, and a horseradish peroxidase-conjugated antibody (Proteintech) at 1:3,000 was applied for another 2 h as the secondary antibody. Beta-actin (Proteintech) was used as an internal control in this assay, and it was used according to the recommended dilution.
The blots were detected using Amersham ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK).
3
Detection of Listeriolysin O in Listeria
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To detect secreted LLO, 20 μl of bacterial culture supernatant was subjected to SDS-PAGE, and the protein was transferred to polyvinylidene fluoride (PVDF) membranes (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The membranes were probed with a primary rabbit anti-LLO antibody and secondary horseradish peroxidase-conjugated anti-rabbit antiserum. Western blot detection was performed using Amersham ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, United Kingdom) and a chemiluminescence imaging system.
To detect surface-associated and internal LLO, bacterial pellets from 300 ml of culture were resuspended in 40 ml of PBS and adjusted to their final concentrations with 10 mM vanadate, 1 mM dithiothreitol (DTT), and 1× proteinase inhibitor mix (Roche Diagnostics), and the cells were then lysed by sonication. The lysates were subjected to SDS-PAGE and subsequent Western blotting, as described above.
To detect surface-associated and internal LLO, bacterial pellets from 300 ml of culture were resuspended in 40 ml of PBS and adjusted to their final concentrations with 10 mM vanadate, 1 mM dithiothreitol (DTT), and 1× proteinase inhibitor mix (Roche Diagnostics), and the cells were then lysed by sonication. The lysates were subjected to SDS-PAGE and subsequent Western blotting, as described above.
4
Western Blot Analysis of Protein Lysates
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Cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethane sulfonyl fluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, and 0.5% NP-40) [11 (link),12 (link)]. For denaturing conditions, their supernatants were denatured in a denaturing sample buffer (Nacalai Tesque). The samples were separated into 8 to 12% sodium dodecyl sulfate–polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred to PVDF membranes (Fujifilm), blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by peroxidase-conjugated secondary antibodies (MBL, Tokyo, Japan). A CanoScan LiDE400 image scanner (Canon, Tokyo, Japan) captured the peroxidase-reactive bands on X-ray films (Fujifilm). Quantified immunoreactive bands were compared with the control’s immunoreactive bands, which were set to 100%, using Image J software (NIH, Bethesda, MD, USA).
5
Protein Extraction and Immunoblotting Protocol
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Following the induction of differentiation for 2 days, cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, 1 μg/mL leupeptin, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, and 0.5% NP-40) to be collected by centrifuge as cell lysates. For denatured conditions, their supernatants were denatured sample buffer (Nacalai Tesque). The samples were separated on pre-made sodium dodecylsulfate–polyacrylamide gels (Nacalai Tesque). The electrophoretically separated proteins were transferred to PVDF membranes (Fujifilm, Tokyo, Japan) sandwiched between filter papers, blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by peroxidase-conjugated secondary antibodies. The Canoscan LiDE400 image scanner (Canon, Tokyo, Japan)-captured, peroxidase-reactive bands on X-ray films (Fujifilm) were analyzed using UN-SCAN-IT software (Silk Scientific, Orem, UT, USA). We performed some sets of experiments in immunoblotting studies and quantified other immunoreactive bands with the control’s immunoreactive band as 100% with NIH ImageJ software (Bethesda, MD, USA).
6
Cell Lysis and Protein Analysis Protocol
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Cells were lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 20 mM MgCl 2 , 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, 1 µg/ml of leupeptin, 1 mM EDTA, 1 mM Na 3 VO 4 , 10 mM NaF, and 0.5% NP-40) and collected by centrifugation as cell lysates [27, 28] . Their supernatants were denatured with a sample buffer (Nacalai Tesque). The samples were separated on pre-made sodium dodecylsulfate-polyacrylamide gels (Nacalai Tesque) [27, 28] . The electrophoretically separated proteins were transferred to PVDF membranes (Fujifilm) sandwiched between filter papers (Fujifilm), blocked with Blocking One (Nacalai Tesque), and immunoblotted using primary antibodies, followed by peroxidase-conjugated secondary antibodies. A CanoScan LiDE400 image scanner (Canon, Tokyo, Japan) captured the peroxidasereactive bands on X-ray films (Fujifilm). They were digitally scanned using UN-SCAN-IT software (Silk Scientific, Orem, UT, USA). Quantified immunoreactive bands (3 blots) were compared with the control's immunoreactive bands, which were set to 100%, using Image J software (NIH, Bethesda, MD, USA).
7
Subcellular Fractionation and Western Blot Analysis
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Western blot analyses were performed as previously described [19 (link)]. The subcellular fractions (cytosolic and nuclear fractions) were separated using a nuclear/cytosolic fractionation kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Protein samples containing 20 μg of total protein were separated via electrophoresis with 4–15% sodium dodecyl sulfate (SDS)–polyacrylamide gels, after which they were transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After transfer, PVDF membrane was blocked with 5% bovine serum albumin (Fujifilm) in TBS containing 0.1% Tween-20 at room temperature for 1 h. Immunoblotting was then performed using primary antibodies against Nrf2 (1:1000, Proteintech, Rosment, IL, USA), GAPDH (1:5000, Cell Signaling Technology, Danvers, MA, USA), and histone H3 (1:2000, Cell Signaling Technology). GAPDH and histone H3 were used as internal standards. Horseradish peroxidase conjugated secondary antibody was used to detect immunoreactivity (Amersham Pharmacia Biotech, Piscataway, NJ, USA), which was visualized using enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech) and RX-U Fuji X-ray film (Fujifilm).
8
Immunoblotting of Cell Lysate Proteins
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Collected cells were lysed in lysis buffer [36 (link),37 (link),38 (link)]. Their centrifugally collected supernatants (20 mg per sample) were denatured in sample buffers (Fujifilm). They were separated on a sodium dodecylsulfate (SDS) polyacrylamide gel. The cell lysate-derived proteins or the resultant precipitates were separated using polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Fujifilm). They were blocked with Blocking One (Nacalai Tesque) and immunoblotted using the respective primary antibodies and peroxidase enzyme–conjugated secondary antibodies.
Enzymatically reactive bands were detected using CanoScan LIDE400 (Canon, Tokyo, Japan) and scanned using CanoScan software (ver. 2023, Canon). The blots shown in the figures are representative of 3 blots. We performed multiple sets of experiments and quantified other bands with one control’s band as 100% using Image J software (ver. Java 8,https://imagej.nih.gov/ accessed on 5 December 2023).
Enzymatically reactive bands were detected using CanoScan LIDE400 (Canon, Tokyo, Japan) and scanned using CanoScan software (ver. 2023, Canon). The blots shown in the figures are representative of 3 blots. We performed multiple sets of experiments and quantified other bands with one control’s band as 100% using Image J software (ver. Java 8,
9
Investigating Anticancer Signaling Pathways
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T24 and 5637 cells were cultured in six-well plates and exposed to various concentrations of MLN4924 (0, 0.1, 0.3, 1 μM), with or without celecoxib (60 μM) for 24 h. Then, the cells were harvested at 80% confluency with a cell scraper and lysed in RIPA lysis buffer (Solarbio) with 1-mM phenylmethylsulfonyl fluoride (PMSF) and 1%-mM phosphatase inhibitors (Solarbio) on ice for 30 min. After measuring protein concentration by the Pierce BCA protein Assay kit (Thermo, USA), the samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). After blocking with 5% skim milk for 1 h (Solarbio), the membranes were incubated with primary antibodies AKT, phospho-AKT (S473), p44/42MAPK (ERK1/2), phospho-p44/42MAPK (ERK1/2), PARP, Bcl2, BAX, N-cadherin, E-cadherin, vimentin, and β-actin (CST, Danvers, MA, USA) at 4°C overnight. Then, the membranes were washed with tris buffered saline tween (TBST, Solarbio) three times (10 min each) and incubated with horseradish peroxidase-conjugated secondary antibodies to IgG (CST) for 1 h at room temperature. The PVDF membrane was quantified using LAS-4000 Mini (Fuji Film, Japan).
10
GST Fusion Protein Binding Assay
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An 35S-labeled protein was prepared using a TnT® Quick Coupled Transcription/Translation System (Promega, Madison, WI, USA), following the manufacturer's instructions. The GST fusion proteins were expressed in Escherichia coli BL21 DE3 and immobilized on Glutathione-Sepharose 4B beads (Cytiva, Tokyo, Japan). The beads were mixed with 35S-labeled protein and incubated for 2 h at 4°C. The beads were subsequently washed five times with wash buffer (20 mM Tris pH 8.0, 2.5 mM MgCl2, 100 mM KCl, 5% glycerol, and 0.1% Tween20), resuspended in sample buffer, analyzed by SDS-PAGE, and transferred to a Poly vinylidene fluoride (PVDF) membrane (Millipore, Billerica, Massachusetts, United States). The GST fusion proteins were detected by Coomaisse brilliant blue staining. The PVDF membrane was subsequently dried and exposed to a BAS-IIIS imaging plate (Fujifilm, Tokyo, Japan) for 3 h. The 35S-labeled proteins were detected using a BAS2000 bio-imaging analyzer (Fujifilm).
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