For Western blot analysis, HeLa cells were maintained in DMEM supplemented with 10% FBS. Cells were harvested 24 h after ROT and AA treatment. After that, the cells were re-suspended in cell lysate buffer [50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% Nonidet P-40, 0.1% deoxycholate, and leupeptin, aprotinin, and 4-(2-aminoethyl) benzenesulfonylfluoride (10 mg/mL each)]. Samples were sonicated and then centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was then placed into a fresh centrifuge tube, protein sample buffer was added, and the sample was heated to 95 °C for 5 min. Proteins were separated by 10–12% SDS/PAGE, transferred to Hybond PVDF membranes (Amersham, GE Healthcare, Velizy-Villacoublay, France), blocked with 5% milk in PBS with 0.05% Tween 20 (PBS/Tween) for 1 h, washed with PBS/Tween 20 three times for 10 min each, and incubated with the relevant antibodies. The blots were washed with PBS/Tween 20 three times for 10 min each, and then HRP-conjugated antibody was used as the secondary antibody for an additional 1 h.
Hybond pvdf membrane
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Germany
Hybond PVDF membrane is a type of polyvinylidene difluoride (PVDF) membrane used in various laboratory applications. It is a durable, chemically resistant material that can be used for protein transfer and detection in techniques such as Western blotting.
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Lab products found in correlation
Ecl kit, by GE Healthcare (3 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
M per, by Thermo Fisher Scientific (2 mentions)
Tgx gel, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Ecl chemiluminescence kit, by PerkinElmer (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti ha, by BioLegend (2 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Sab2702197, by Merck Group (1 mentions)
G6532, by Merck Group (1 mentions)
Amersham ecl prime reagent, by GE Healthcare (1 mentions)
F1804, by Merck Group (1 mentions)
Las4000, by GE Healthcare (1 mentions)
Rabbit anti lamin b1, by Abcam (1 mentions)
Rabbit anti hif 1α d2u3t, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
38 protocols using hybond pvdf membrane
1
Western Blot Analysis of HeLa Cells
2
Western Blotting Protocol for Protein Analysis
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Western blotting was performed following a previously published protocol (Miyakoshi et al., 2019 (link)). Briefly, whole‐cell samples in 1× Laemmli sample buffer (Bio‐Rad) were separated on 10% or 12% TGX gels (Bio‐Rad). Proteins were transferred onto Hybond PVDF membranes (GE Healthcare), and membranes were blocked for 10 min in Bullet Blocking One buffer (Nacalai Tesque) and were incubated overnight at 4℃ with monoclonal anti‐FLAG (Sigma–Aldrich #F1804; 1:5,000), monoclonal anti‐GFP (Sigma–Aldrich SAB2702197; 1:5,000), and polyclonal anti‐GroEL (Sigma–Aldrich #G6532; 1:10,000) antibodies diluted in Bullet Blocking One buffer. Membranes were washed three times for 15 min in 1 × TBST buffer at RT. Then membranes were incubated for 1 hr at RT with secondary antimouse or antirabbit HRP‐linked antibodies (Cell Signaling Technology #7076 or #7074; 1:5,000) diluted in Bullet Blocking One buffer and were washed three times for 15 min in 1 × TBST buffer. Chemiluminescent signals were developed using Amersham ECL Prime reagents (GE Healthcare), visualized on LAS4000 (GE Healthcare), and quantified using Image Quant TL software.
3
Western Blot Analysis of Protein Samples
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Total cell lysates were obtained using Laemmli or RIPA buffer, as previously described [30 (link),36 (link)]. Proteins were separated by SDS-PAGE and transferred onto Hybond™ PVDF membranes (GE Healthcare, IL, USA) by electroblotting. Infrared imaging (Odyssey, Li-Cor Bioscience, Lincoln, NE, USA) was used to detect protein bands. Images were recorded as TIFF files for quantification with ImageJ software. Antibodies used are listed in Supplementary Table S2 .
4
Quantifying Nuclear HIF-1α Expression
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Nuclear proteins were extracted from hypoxic and normoxic cells using a NE-PER nuclear and cytoplasmic extraction kit (Cat# 78833, Thermo-Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Equal amounts of nuclear protein were electrophoresed in 10% SDS-polyacrylamide gels and transferred to hybond-PVDF membranes (Cat# 23225, GE Healthcare Life Sciences, Pittsburgh, PA, USA). The primary antibodies used in this study were: rabbit anti-Lamin B1 (Cat# ab133741, Abcam, Cambridge, MA, USA) and rabbit anti-HIF-1α (D2U3T) (Cat# 14179, Cell Signaling Technology, Danvers, MA, USA). Next, the membranes were incubated with anti-rabbit Ig, peroxidase-linked, species-specific whole antibody (Cat# 31460, Thermo-Fisher Scientific). ECL reagents were used to detect the signals according to the manufacturer’s instructions (Cat# RPN3244, GE Healthcare Life Sciences, Pittsburgh, PA, USA). All films were scanned with an Epson Expression 1680 optical scanner (Epson America Inc., Long Beach, CA, USA). Densitometry analysis of bands was performed using an open-source, public domain software package (ImageJ v1.47).
5
Western Blot Analysis of 3xHA-HsfA1a
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Seedlings were homogenized in extraction buffer (150 mM Tris–HCl, pH 7.5, 6 M urea, 2% SDS, and 5% µ-mercaptoethanol). Samples were boiled, and cell debris was removed by centrifugation at 18,000×g at 4°C for 10 min. The supernatants were resolved on 12% SDS polyacrylamide gel electrophoresis, transferred to Hybond PVDF membranes (GE Healthcare) and subjected to Western blot analysis. For detection 3xHA-tagged HsfA1a, horseradish peroxidase conjugated antibody (Roche, 3F10) was used. The proteins were visualized by chemiluminescence (ECL kit; GE Healthcare) according to the manufacturer's instructions.
6
Western Blot Analysis of Heat Stress Proteins
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Seedlings were homogenized in extraction buffer (150 mM Tris–HCl, pH 7.5, 6 M urea, 2% SDS and 5% μ-mercaptoethanol), boiled for 5 min and cell debris removed by centrifugation at 18 000 × g at 4°C for 10 min. The supernatants were resolved on 12% SDS-PAGE, transferred to Hybond PVDF membranes (GE Healthcare) and subjected to western blot analysis. Antibodies used for detection: for 3xHA-tagged HsfA1a, HRP-conjugated antibody (3F10, Roche); for GSy tag (52 (link)), anti-GFP antibody (MA5-15256, Thermo Fisher); for sHSP-CI, anti-sHSP17.6 antibody (AS07 254, Agrisera), anti-HSP70 antibody (AS08 371, Agrisera), anti-HSP90-1 antibody (AS08 346, Agrisera), anti-HSP101 (AS07 253, Agrisera), anti-Ub antibody (U5379, Sigma-Aldrich) and anti-SUMO1 antibody (AS08 308, Agrisera); as secondary antibody, we used monoclonal HRP-conjugated anti-mouse (A4416, Sigma-Aldrich) or anti-rabbit (A6154, Sigma-Aldrich). The proteins were visualized by chemiluminescence (ECL kit; GE Healthcare) and quantified by Image Lab 5.1 (Bio-Rad); protein signals have been normalized to Rubisco large subunit (RbcL), having a slow turnover rate (67 (link)).
7
Western Blot Protein Analysis Protocol
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Cell lysates were harvested in RIPA buffer (Sigma) containing protease inhibitors (Sigma). The protein content was determined using the Pierce™ BCA Protein Assay Kit (Thermo-Fisher Scientific) and subsequently normalised, such that 10 or 20 µg of total protein could be loaded, per lane, onto SDS-PAGE gels. Proteins were resolved by SDS-PAGE and then transferred to Hybond PVDF membranes (GE Healthcare). Membranes were incubated for 1 h in PBS, 0.2% Tween-20, 5% skimmed milk powder, prior to an overnight incubation with the primary antibody diluted in the same blocking buffer. Membranes were washed in PBS containing 0.2% Tween-20, before incubating for 1 h with the HRP-conjugated secondary antibody. Membranes were washed prior to detection of chemiluminescence by incubating the membrane with LumiGLO (Cell Signalling Technology) for 1 min. Membranes were then exposed to X-ray film (Fujifilm) and developed manually using developer and fixer from AGFA.
8
Protein Expression Analysis of MCF-7 and MDA-MB-453 Cells
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The whole‐cell proteins from MCF‐7 and MDA‐MB‐453 cells were extracted using M‐PER mammalian protein extraction reagent (Thermo Fisher Scientific Pierce Biotechnology, Rockford, IL). The lysate proteins (10 μg) were subjected to SDS–PAGE (10% acrylamide gel). Following SDS–PAGE, the proteins were transferred onto Hybond PVDF membranes (GE Healthcare, Buckinghamshire, UK). Primary antibody used was anti‐TACC2 antibody (Gene Tex), ER (6F11, Novocastra, Newcastle upon Tyne, UK) and AR (AR441, DAKO). In addition, anti‐β‐actin (AC‐15, Sigma‐Aldrich) antibody was used as an internal control. Antibody‐protein complexes on the blots were detected using ECL‐Plus Western Blotting Detection Reagents (GE Healthcare), and the protein bands were visualized using ChemiDoc™ XRS+ system (Bio‐Rad Laboratories, Inc., Hercules, CA).
9
Mitochondrial Protein Extraction and Analysis
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The mitochondrial protein from MCF10DCIS.com cells was extracted according to a previous report20 and M‐PER (Thermo Fisher Scientific Pierce Biotechnology, Rockford, IL, USA). The lysate proteins (2 μg) were subjected to SDS–PAGE (10% acrylamide gel) and transferred onto Hybond PVDF membranes (GE Healthcare, Little Chalfont, UK). The primary anti‐CYC1 antibody used was the same as that in the immunohistochemistry. Antimitochondrial voltage‐dependent anion channel 1 antibody (sc‐390996; Santa Cruz Biotechnology, Dallas, TX, USA) was also used as an internal control for mitochondrial protein.21
10
Western Blotting: Protein Analysis
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Western blotting was done as previously described (61 (link)) using Hybond PVDF membranes (GE Healthcare) and the following antibodies: GINS3 (Abcam, AB177515), GINS1 (Abcam, AB183524), FLAG (MilliporeSigma, F1804), V5 (Bio-Rad, MCA1360GA), HA (12CA5; Abcam, AB1424), GAPDH (Santa Cruz Biotechnology Inc., sc-25778), and actin (Abcam, AB1801). Bands of interest were quantified by densitometry using ImageJ software (NIH).
For details of immunoprecipitation and immunolabeling procedures, see Supplemental Methods.
For details of immunoprecipitation and immunolabeling procedures, see Supplemental Methods.
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