Cells were lysed using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 M EDTA, 1% Triton-X, and 0.1% sodium dodecyl sulfate (SDS)) supplemented with 10 mM NaF, 2 mM Na3VO4, and a complete Mini Protease Inhibitor Cocktail (Roche Diagnostics). Protein content was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Proteins were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in TBS-t containing 5% skimmed milk for 1 h at room temperature before incubating with the primary antibodies overnight at 4 °C. The primary antibodies used and their working conditions are summarized in Table S1 . Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (GE Healthcare, Buckinghamshire, England, UK; 1:10,000, TBSt-milk, room temperature) as the secondary antibody for 1 h. The membranes were developed using the ECL Prime Western Blotting Detection System (GE Healthcare) and luminescence was captured and quantified using an imaging system (ChemiDoc Image Lab, Bio-Rad Laboratories).
Polyvinylidene difluoride membrane
Manufactured by Bio-Rad
Sourced in United States, Germany, Japan, United Kingdom, China, Canada, Italy, Ireland
Polyvinylidene difluoride (PVDF) membranes are a type of laboratory equipment used for protein and nucleic acid transfer and detection in various techniques, such as Western blotting and Northern blotting. PVDF membranes offer high binding capacity for proteins and nucleic acids, and are commonly used for their durability, chemical resistance, and low background signal.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (74 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (53 mentions)
Ripa buffer, by Thermo Fisher Scientific (40 mentions)
Protease inhibitor cocktail, by Merck Group (38 mentions)
β actin, by Cell Signaling Technology (38 mentions)
β actin, by Merck Group (36 mentions)
Ripa lysis buffer, by Beyotime (34 mentions)
Bca protein assay kit, by Beyotime (31 mentions)
Image lab software, by Bio-Rad (30 mentions)
Clarity western ecl substrate, by Bio-Rad (28 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (28 mentions)
Chemidoc xrs system, by Bio-Rad (24 mentions)
Chemidoc touch imaging system, by Bio-Rad (21 mentions)
Bca protein assay, by Thermo Fisher Scientific (21 mentions)
Quantity one software, by Bio-Rad (20 mentions)
Gapdh, by Cell Signaling Technology (19 mentions)
Protease inhibitor cocktail, by Roche (19 mentions)
Chemidoc mp imaging system, by Bio-Rad (19 mentions)
Cell lysis buffer, by Cell Signaling Technology (19 mentions)
Chemidoc xrs imaging system, by Bio-Rad (19 mentions)
1 272 protocols using polyvinylidene difluoride membrane
1
Western Blot Protein Expression Analysis
2
Collagen and 7f Modulate Cell Signaling
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Pan02, BMF-A3, and CT1A-C11 cells
were cultured in DMEM (Corning) containing 10% fetal bovine serum
(FBS) and maintained at 37 °C in a humidified incubator with
5% CO2 and 95% air. For the collagen and7f treatment, cells were cultured in 10 mL of complete media with 50
μg/mL collagen and indicated concentration of7f for 8 or 18 h. Cells were lysed, supernatants were recovered by
centrifugation at 13 000 rpm, protein concentration was measured,
and equal amounts of total protein were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Proteins were transferred
to poly(vinylidene difluoride) membranes (Bio-Rad, Hercules, CA) followed
by blockade for 1 h in 5% bovine serum albumin in TBS-T. Membranes
were incubated overnight at 4 °C with primary antibody phospho-DDR1
(Tyr792, Cell Signaling #11994), DDR1 (Santa Cruz SC-532), phospho-PYK2
(Tyr402, Cell Signaling #3291), PYK2 (Cell Signaling #3292), PEAK1
(Millipore 09-274), phospho-PEAK1 (Tyr665, Millipore #ABT52), E-cadherin
(24E10, Cell Signaling #3195), N-cadherin (13A9, Cell Signaling #3195),
and TUBULIN α (Biorad, MCA77D800). Membranes were incubated
with the corresponding horseradish peroxidase-conjugated secondary
antibody (Pierce Biotechnologies, Rockford, IL) for 1 h. Specific
bands were detected using the enhanced chemiluminescence reagent (ECL,
PerkinElmer Life Sciences, Boston, MA) on an autoradiographic film.
were cultured in DMEM (Corning) containing 10% fetal bovine serum
(FBS) and maintained at 37 °C in a humidified incubator with
5% CO2 and 95% air. For the collagen and
μg/mL collagen and indicated concentration of
centrifugation at 13 000 rpm, protein concentration was measured,
and equal amounts of total protein were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Proteins were transferred
to poly(vinylidene difluoride) membranes (Bio-Rad, Hercules, CA) followed
by blockade for 1 h in 5% bovine serum albumin in TBS-T. Membranes
were incubated overnight at 4 °C with primary antibody phospho-DDR1
(Tyr792, Cell Signaling #11994), DDR1 (Santa Cruz SC-532), phospho-PYK2
(Tyr402, Cell Signaling #3291), PYK2 (Cell Signaling #3292), PEAK1
(Millipore 09-274), phospho-PEAK1 (Tyr665, Millipore #ABT52), E-cadherin
(24E10, Cell Signaling #3195), N-cadherin (13A9, Cell Signaling #3195),
and TUBULIN α (Biorad, MCA77D800). Membranes were incubated
with the corresponding horseradish peroxidase-conjugated secondary
antibody (Pierce Biotechnologies, Rockford, IL) for 1 h. Specific
bands were detected using the enhanced chemiluminescence reagent (ECL,
PerkinElmer Life Sciences, Boston, MA) on an autoradiographic film.
3
Nuclear Protein Extraction and Analysis
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Nuclear proteins were collected from HM-1-B7-H3 KO cells, ID8-B7-H3 KO cells or their control cells, and OVCAR3-sh-B7-H3 and OVCA420-sh-B7-H3 or their control cells using NE-PER nuclear and cytoplasmic extraction reagents (catalog no. 78833, Thermo Fisher Scientific). Protein (20 μg) of HM-1-B7-H3 KO cells or their control cells, and 10 μg protein of the other cells were loaded onto 8% acrylamide gels. Proteins were subsequently separated using SDS-PAGE and transferred onto polyvinylidene difluoride membranes (catalog no. 1620177, Bio-Rad). After blocking for 1 hour with Blocking One-P (catalog no. 05999-84, Nacalai Tesque), the membranes were then immunoblotted with the antibodies listed in Supplementary Table S2 at the indicated dilutions. The membranes were incubated with the primary antibody overnight at 4°C and with the secondary antibody for 1 hour at room temperature. Bands were visualized using ChemiDoc XRS+ Systems (Bio-Rad). Signals were quantified using Image Lab 2.0 (RRID: SCR_014210, Bio-Rad).
4
Immunoblotting for Xenopus ADHFe1 Protein
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A rabbit polyclonal antibody raised against amino acids 110-335 of the human ADHFe1 (PA5-31416; Thermo Fisher Scientific) was used to detect the Xenopus laevis protein. Protein lysates for Western blots were prepared by homogenizing embryos or HEK293 cells in ice-cold lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with protease inhibitors (1 mM phenylmethyl sulfonyl fluoride, 1 mM pepstatin, and 10 mg/ml aprotinin). Homogenates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C. Sodium dodecyl sulfate (SDS) sample buffer was added to the cleared lysate and boiled for 4 min before separation by SDS-polyacrylamide gel electrophoresis. Variability in cell densities was minimized by normalization to total protein concentration measured with the protein assay kit (Bio-Rad). Proteins were blotted to polyvinylidene difluoride membranes (Bio-Rad), and blots were blocked in 5% milk in TBS + 0.1% Tween and probed with anti-ADHFe1 antibody (1:10000) for 1 hr at room temperature and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20000; A0545, Sigma) as a second antibody. Immunoreactive proteins were detected using a chemiluminescence kit (Biological Industries) according to the manufacturer's protocol.
5
Protein Extraction and Western Blot Analysis in GVHD Rat Tissues
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Total protein was extracted from spleen and tongue specimens in control or GVHD rats using ice-cold cell lysis buffer (20 m M Tris–HCl, pH 7.5; 150 mMNaCl; 1 mM ethylenediaminetetraacetic acid [EDTA]; 1 mM Na2EDTA; 1 mM ethylene glycol tetraacetic acid; 1% [v/v] Triton-X 100; 2.5 mM sodium pyrophosphate; 1 mMβ-glycerophosphate; 1 mM Na3VO4; and 1 μg/ml leupeptin and phenylmethylsulfonyl fluoride). Equal amounts of protein (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% separating gel). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Tokyo, Japan). The blots were blocked with 1% casein in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. Primary Abs against LMAN2 (described earlier) and β-actin (Sigma-Aldrich) were used. Membranes were washed in TBS-T and incubated with secondary horseradish peroxidase-labeled Ab for 1 h at room temperature. Bound Ab complexes were detected by enhanced chemiluminescence (Bio-RadLaboratories).
6
Western Blot Analysis of DNA Repair Proteins
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B cells were lysed in Laemmli buffer. Cell extracts containing equal amounts of protein (50-100 μg) were fractionated through SDS-PAGE (6%). The fractionated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) overnight (30 V/90 mA) at 4 °C. After blocking and overnight incubation at 4 °C with anti-AID Ab (H-80, Santa Cruz), anti-Ku70 Ab (A0883, Abclonal), anti-Ku86 Ab (A5862, Abclonal), anti-Rad52 Ab (H-300, Santa Cruz Biotechnology), anti-phospho-Rad52 Ab (Y408472, Applied Biological Materials Inc.) or anti-β-Actin mAb (clone 2F1-1, 643802, BioLegend), the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. After washing with TBS-Tween 20 (0.05%), bound HRP-conjugated antibodies were detected using Western Lightning Plus-ECL reagents (PerkinElmer Life and Analytical Sciences).
7
Western Blot Analysis of COX-IV
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Equal amount of supernatant from the pellet of cell culture medium were loaded onto 4–12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad laboratories, Hercules, CA, USA). The membranes were incubated with blocking buffer containing 0.1% Tween-20 and 5% skimmed milk and probed with mouse anti-COX-IV antibody (Molecular Probes) or β-actin antibody (Sigma-Aldrich) as a loading control. Following incubation with horseradish peroxidase-conjugated anti-mouse secondary antibody (DAKOCytomation, Glostrup, Denmark), the bound antibody was visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
8
Western Blot Analysis of Stroke Biomarkers
9
Western Blot Analysis of Adipogenic Markers
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Differentiated 3T3-L1 cells were lysed with PRO-PREP Protein Extraction Solution (iNtRON, Korea) in ice for 1h, followed by centrifugation at 10,000 ×g for 10 min at 4°C. Proteins (30 μg) were subjected to Tris-Glycine gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Bio-Rad, USA). The membranes were incubated with primary antibodies including GAPDH, C/EBPα, PPARγ, and SREBP-1c (1:1000, Cell Signaling Technology, USA), followed by incubation with anti-rabbit secondary antibodies (Cell Signaling Technology). Protein bands were visualized using an enhanced chemiluminescence system (ECL Advance, GE Healthcare, UK). Protein density was calculated using the Image J 1.53 program (NIH, USA).
10
Immunoblotting for TUBB3 Expression
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Anti-E-cadherin, anti-ATP-binding cassette subfamily B, member 1 (ABCB1), anti-class III beta-tubulin (TUBB3) and anti-beta-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA).
Preparation of total cell lysates and immunoblotting was performed as previously described [17 (link)]. Cells were cultured without erlotinib until subconfluency, and media was changed to RPMI with 10% FBS containing DMSO or 1 μM entinostat. After 72 hours, cells were rinsed with phosphate-buffered saline (PBS), lysed in sodium dodecyl sulfate (SDS) buffer and homogenized. Approximately 30 μg of total cell lysate protein was subjected to SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). After blocking with 2.5% nonfat dry milk and 2.5% bovine serum albumin in PBS, membranes were incubated with primary antibodies (1:1000) overnight, washed with PBS, reacted with secondary antibody (1:1000), treated with ECL solution (GE Healthcare, Fairfield, CT). Chemiluminescence was detected by EOS Kiss X6i (Canon, Tokyo, Japan). Expression values of TUBB3 relative to beta-actin were determined using Just TLC software (Sweday, Lund, Sweden).
Preparation of total cell lysates and immunoblotting was performed as previously described [17 (link)]. Cells were cultured without erlotinib until subconfluency, and media was changed to RPMI with 10% FBS containing DMSO or 1 μM entinostat. After 72 hours, cells were rinsed with phosphate-buffered saline (PBS), lysed in sodium dodecyl sulfate (SDS) buffer and homogenized. Approximately 30 μg of total cell lysate protein was subjected to SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). After blocking with 2.5% nonfat dry milk and 2.5% bovine serum albumin in PBS, membranes were incubated with primary antibodies (1:1000) overnight, washed with PBS, reacted with secondary antibody (1:1000), treated with ECL solution (GE Healthcare, Fairfield, CT). Chemiluminescence was detected by EOS Kiss X6i (Canon, Tokyo, Japan). Expression values of TUBB3 relative to beta-actin were determined using Just TLC software (Sweday, Lund, Sweden).
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