Cells were harvested and lysed in cell lysis buffer (Beyotime). Cell lysates were incubated on ice for 15 mins and centrifuged at 12,000 rpm for 10 mins at 4°C. Protein levels were measured by BCA Protein Assay Kit (Thermo Fisher Scientific, San Jose, CA, USA). Boiled samples were loaded equally into SDS-PAGE and transferred onto PDVF membranes (Roche, Basel, Switzerland). After 1 hr blocking in 5% milk at room temperature, membranes were incubated with primary antibodies and rotated overnight at 4°C. After washing with PBS containing 0.1% Tween-20 and incubation of secondary antibodies, ECL Western Blotting Substrate (Thermo Fisher Scientific) was used to detect protein bands. Antibodies used for western blot assay were: PRMT5 (Abcam, USA, ab109451) and GAPDH (Abcam, USA, ab181602).
Pdvf membranes
Manufactured by Roche
Sourced in Switzerland, Germany
PVDF membranes are a type of laboratory equipment used for various applications. They are made of polyvinylidene fluoride (PVDF), a chemical compound with high chemical and thermal stability. PVDF membranes are commonly used for filtration, separation, and purification processes in a wide range of research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Ab109451, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab181602, by Abcam (1 mentions)
Sc 397, by Santa Cruz Biotechnology (1 mentions)
Sc 6243, by Santa Cruz Biotechnology (1 mentions)
Ab199728, by Abcam (1 mentions)
Ab54576, by Abcam (1 mentions)
Ab184796, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ecl prime western blotting detection reagent, by Merck Group (1 mentions)
3 methyladenine, by Merck Group (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc early apoptosis detection kit, by Cell Signaling Technology (1 mentions)
Laemmli buffer 4x, by Bio-Rad (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
7 protocols using pdvf membranes
1
Western Blot Analysis of PRMT5
2
UV-Induced DNA Damage Response in Cells
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Confluent 60 mm dishes containing CT-26, 4T1 or Vero cells were exposed to UV irradiation at 4 J/m2 for 0, 2, 5 and 10 s, and 16 h after irradiation cells were washed twice with ice-cold Dulbecco’s PBS 1×(PBS), harvested and lysed in a hypotonic buffer (10 mM Tris-HCl pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 2 mM DTT, 1 mM Pefabloc, 2 mM sodium vanadate, 4 µg/mL pepstatin, 4 µg/mL leupeptin, and 4 µg/mL aprotinin) for 30 min at 4°C, and centrifuged at 13,000 g for 10 min at 4°C to remove debris. Equivalent amounts of protein were separated by SDS-PAGE and wet-electrotransferred onto PDVF membranes (Roche). Membranes were blocked for 1 h at room temperature with blocking buffer (PBS, 0.1% Tween 20, and 5% nonfat dry milk) and proteins were detected with antibodies against p21 (sc-397, Santa Cruz), and p53 (sc-6243, Santa Cruz). Primary antibodies were diluted 1∶200 in dilution buffer (PBS, 0.1% Tween 20, and 5% nonfat dry milk) and blots were incubated overnight, followed by 1 h incubation with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibody (Santa Cruz) (1∶10,000). Peroxidase activity was revealed by enhanced chemiluminescence (Pierce). GAPDH was used as loading control.
3
Western Blot Analysis of Cell Cycle Regulators
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Cells were resuspended in RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Sodium deoxycholate, 0.1% SDS, 25 mM Tris pH 7.4). Cell lysates were incubated on ice for 20 mins and supernatants were collected after high-speed centrifuge at 4°C. Protein concentration was measured by Bradford assay. Samples were loaded evenly into SDS-PAGE and transferred onto PDVF membranes (Roche). Membranes were blocked in 5% skim milk at room temperature and then incubated with primary antibodies overnight at 4°C. After washing with PBS-T (PBS solution, 0.1% Tween-20) and incubation of secondary antibodies, membranes were incubated with ECL Western Blotting Substrate (Thermo Fisher Scientific) and protein bands were developed in dark room. Antibodies used for Western blot assay were: CCND1 (abcam, ab16663), pRb (abcam, ab184796), CDK4 (abcam, ab199728), CDK6 (abcam, ab54576), GAPDH (abcam, ab9485).
4
Protein Expression Analysis by Western Blot
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Cells and tumor tissue were lysed with RIPA buffer. The protein concentration was determined by a BCA protein assay kit. SDS-PAGE gels were used to electrophorese the total protein, which was then transferred to PDVF membranes (Roche, Penzberg, Germany). The membranes were blocked with 5% skimmed milk dissolved in TBST for 2 hours and incubated overnight at 4°C with the primary antibody. After washing three times with PBST, membranes were incubated with secondary antibody for 2 h at room temperature. The membranes were washed with TBST three times, and a ChemiDoc XRS System (Bio-Rad, CA, USA) was used to detect the expression of proteins with an enhanced chemiluminescence (ECL) kit (NCM Biotech, Suzhou, China).
5
Comprehensive Cell Viability and Apoptosis Assay Protocol
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MEM (minimum essential medium), DMEM (Dulbecco’s Modified Eagle Medium), FBS (Fetal bovine serum), nonessential amino acids, trypsin-EDTA, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), BSA (bovine serum albumin), 5-(N-ethyl- N-isopropyl)amiloride (EIPA), ECL™ Prime Western Blotting Detection Reagent, and 3-methyladenine were from Sigma-Aldrich/Merck. DMEM FluoroBrite, lysotracker red, and Lucifer yellow (LY) were from ThermoFisher Scientific. z-VAD and fumonisin B1 (FB1) were from Enzo Life Sciences. Annexin V-FITC early apoptosis detection kit was from Cell Signaling. Laemmli buffer 4x and 30% acrylamide/Bis 37.5:1 were from Bio-Rad. lysotracker red and PDVF membranes were from Roche. Antibodies: β-actin (mouse) was from Sigma; LC3II (rabbit) from MBL; Caspase 3 (rabbit), Akt (rabbit), pAkt (rabbit), AMPK (rabbit), pAMPK (rabbit), S6 (rabbit), and pS6 (rabbit) were from Cell Signaling. HRP-secondary antibody goat anti-Mouse IgG was from Thermo Fisher Scientific (Barcelona, Spain). HRP-secondary antibody goat anti-Rabbit was from Sigma (Fontenay-sous-Bois, France).
6
Protein Expression Analysis in Pancreatic Cancer
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Pancreatic cancer tissues or PANC‐1 cells were lysed in RIPA buffer (Solarbio) containing 1% PMSF. A BCA kit (Solarbio) was used to quantify the protein concentration. Subsequently, the proteins were separated by SDS‐PAGE and transferred onto PDVF membranes (Roche). The membranes were blocked with 5% nonfat milk, incubated with primary antibodies against NT‐3 (ab16640, Abcam), TrkC (ab227289, Abcam), Vimentin (ab8978, Abcam), N‐Cadherin (ab76011, Abcam), E‐Cadherin (ab1416, Abcam), and GAPDH (ab8245, Abcam) at 4°C overnight, and then incubated with a horseradish peroxidase‐conjugated secondary antibody. The bands of proteins were visualized using an ECL Plus detection system, and the gray value was normalized to GAPDH.
7
Western Blot Analysis Protocol
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Proteins were resolved by SDS-PAGE on 8, 10, or 12% gels. Proteins were transferred to PDVF membranes (Roche CAT# 03010040001) at 100 V for 1 h. Non-specific binding was blocked with 5% milk or bovine serum albumin in tris-buffered saline + Tween 20 for 1 h. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. Membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies for 1 h, then exposed to chemiluminescent reagent. Membranes were imaged using the ChemiDoc Imaging System (Bio-Rad) and densitometry was performed using Image Lab 6.1 software package (Bio-Rad).
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