Cells or tissue homogenate were lysed with RIPA lysis buffer containing PMSF, protease and phosphatase inhibitor cocktail. The protein concentration of each sample was quantified by BCA protein quantification assay kit (Vazyme, E112-01). Twenty μg of lysate was separated using SDS-PAGE gels and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skim milk in TBST for 1 h at room temperature and probed with indicated primary antibody overnight at 4 °C. The next day, PVDF membranes were incubated with anti-mouse or anti-rabbit antibody for 1 h, after which the membranes were treated with ECL Chemiluminescence Kit (Vazyme, E423) and visualized in Tanon 4600 image system. Antibodies against β-actin (AC038), PARP1 (A0942), Lamin B (A16909), FLAG (AE092), γ-H2AX (Ser139) (AP0099), GAPDH (AC001), HIS (AE028) were from ABclonal, while antibody against PAR (ALX-804-220-R100) was from Enzo Life Sciences.
Pvdf membranes
Manufactured by Vazyme
Sourced in China
PVDF (polyvinylidene fluoride) membranes are a type of lab equipment used for various applications in scientific research and analysis. These membranes are known for their excellent chemical and thermal resistance, as well as their high mechanical strength. PVDF membranes are commonly used in filtration, separation, and detection processes in various fields, such as biochemistry, biotechnology, and analytical chemistry.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Ab205718, by Abcam (3 mentions)
Ripa buffer, by Vazyme (3 mentions)
Ecl kit, by Vazyme (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Detergent compatible bradford protein quantification kit, by Vazyme (2 mentions)
Alx 804 220 r100, by Enzo Life Sciences (1 mentions)
Ecl chemiluminescence kit, by Vazyme (1 mentions)
E cadherin, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab2213, by Abcam (1 mentions)
Ab126250, by Abcam (1 mentions)
Ab52771, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab40776, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
5 protocols using pvdf membranes
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of Cellular Proteins
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Proteins from cells were isolated using RIPA buffer (Vazyme) and the concentration was checked by Detergent Compatible Bradford Protein Quantification Kit (Vazyme). Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to segregate proteins and then the proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes (Vazyme). After being blocked with 5% skimmed milk (Vazyme) and washed by phosphate-buffered saline (PBS), the membranes were incubated with the primary antibodies: E-cadherin (1:3000, ab40772, Abcam, Cambridge, United Kingdom), Vimentin (1:2500, ab92547, Abcam), YAP1 (1:3000, ab52771, Abcam), C3aR (1:1000, ab126250, Abcam), ICAM-1 (1:1000, ab2213, Abcam), or β-actin (1:2500, ab8227, Abcam) overnight. After being rewashed with PBS, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad, Richmond, CA, USA) after being treated with ECL kit (Vazyme).
3
Protein Expression Analysis via Western Blot
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Proteins were isolated using RIPA buffer (Vazyme) and segregated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto the polyvinylidene difluoride (PVDF) membranes (Vazyme) and the membranes were blocked with 5% skimmed milk (Vazyme). After being washed by phosphate-buffered saline (PBS), the membranes were incubated with the primary antibodies: Cyclin D1 (1:1000, ab16663, Abcam, Cambridge, United Kingdom), B-cell lymphoma 2 associated X (Bax) (1:3000, ab32503, Abcam), Cleaved-caspase-3 (Cleaved-casp-3) (1:1000, ab2302, Abcam), phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (1:2000, ab40776, Abcam), protein kinase B (AKT) (1:1000, ab8805, Abcam) and phosphorylated AKT (p-AKT) (1:1000, ab38449, Abcam) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam) overnight. After being rewashed, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad) after being treated with ECL kit (Vazyme).
4
Western Blot Analysis of Cell Lysates
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Six-well plate cell samples (2×106 cells/well) were lysed in cell lysis buffer (Beyotime, China) containing phenylmethylsulfonyl fluoride (PMSF) (Beyotime, China) and phosphatase inhibitors. Cell lysates were subjected to 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred by semi-dry blotting onto a polyvinyl difluoride (PVDF) membrane (Merck Millipore, USA). Membranes were blocked with 3% bovine serum albumin (BSA) (Beyotime, China) in TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. After blocking, membranes were probed with the indicated primary antibodies overnight at 4°C. After thorough washing, the membranes were incubated with the corresponding secondary antibodies for 1 h at 37°C. The protein bands were visualized using an enhanced chemiluminescence (ECL) reagent (Vazyme, China), and a chemiluminescence imaging system (0I-X6, China) was used to analyze the PVDF membranes. Densitometry of protein band intensity was performed using ImageJ software from NCBI.
5
Protein Quantification and Western Blot Analysis
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Proteins were isolated by RIPA buffer (Vazyme), and Detergent Compatible Bradford Protein Quantification Kit (Vazyme) was used to determine the concentration of proteins. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins and then the proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes (Vazyme). After being blocked with 5% skimmed milk (Vazyme) and washed by phosphate-buffered saline (PBS), the membranes were incubated with corresponding primary antibodies: MUC19 (1:1000 dilution, ab212621, Abcam, Cambridge, UK), GAPDH (1:2000, ab37168, Abcam), PCNA (1:3000, Abways Technology, Inc., Shanghai, China), cyclin D1 (1:1000, Abways Technology), and antibodies against hexokinase II (HK2) (ab227198, 1:5000, 102 kDa) overnight at 4 °C. Then the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The blots were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad, Richmond, CA, USA) after being incubated with ECL kit (Vazyme).
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