Cells were primed in medium containing DMEM, l -glutamine, pyruvate, nonessential amino acids, dialyzed serum (Life Technologies), and penicillin-streptomycin for 15 min at 37°C. Cells were metabolically labeled in medium containing 50 µCi [35S]methionine/cysteine (Hartmann Analytic). For the negative control, 100 µg/ml cycloheximide was added to the medium. Cells were collected at different time points, lysed in buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, pH 8, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, and protease inhibitor cocktail. The proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (VWR International) that were then exposed to x-ray film.
Polyvinylidene difluoride membrane
Manufactured by Avantor
Sourced in United States
Polyvinylidene difluoride (PVDF) membrane is a type of filtration membrane used in various laboratory applications. It is a synthetic polymer material known for its chemical resistance, mechanical strength, and thermal stability. PVDF membranes are designed to facilitate the separation, isolation, and purification of a wide range of biological and chemical compounds.
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Lab products found in correlation
Horseradish peroxidase tagged anti flag m2 antibodies, by Merck Group (2 mentions)
Sds page, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dialyzed serum, by Thermo Fisher Scientific (1 mentions)
35s methionine cysteine, by Hartmann Analytic (1 mentions)
Trans blot sd transfer cell, by Bio-Rad (1 mentions)
Peroxidase labeled secondary antibody, by Cell Signaling Technology (1 mentions)
Luminata crescendo horseradish peroxidase substrate, by Merck Group (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Ibright cl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
5 protocols using polyvinylidene difluoride membrane
1
Protein Synthesis Kinetics Protocol
2
Western Blot Analysis of DPP3 Protein
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Tissue lysates were diluted in Laemmli's sample buffer, and 20 µg of total protein/lane was subjected to SDS-PAGE using 10% SDS–polyacrylamide gels. The resolved proteins were transferred onto polyvinylidene difluoride membranes (VWR, Radnor, PA, USA) using a Trans-Blot SD transfer cell (Bio-Rad). Following transfer, the membranes were washed with TBS containing 0.01% Tween 20 (TBST) and then blocked in 5% nonfat milk for 1 h at room temperature. The membranes were then incubated overnight with anti-DPP3 rabbit polyclonal antibody (1:1,500; Proteintech Europe, Manchester, UK) in TBST containing 5% nonfat milk at 4 °C. After washing three times for 10 min in TBST, the membranes were incubated with peroxidase-labeled secondary antibody (1:5,000; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The immunoblots were developed using enhanced chemiluminescent Western blotting substrate solution (Pierce–Thermo Fisher Scientific).
3
Quantitative Western Blot Analysis
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Expression level across all variants was assessed by Western blot analysis (Fig. S3 ). About 3 ml of cells were pelleted and resuspended in 300 μl of lysis buffer (50 mM Hepes, pH 8.0, and 50 mM NaCl) with 5 mM β-mercaptoethanol (βME). The cells were sonicated and centrifuged at 21,000g for 10 min before collecting the supernatant. Total protein concentration was determined by bicinchoninic acid assay (Pierce). About 120 μl of lysates were mixed with 40 μl of 4× LDS sample buffer (Novex, Life Technologies) with βME and boiled at 98 °C for 3 min. For each FtsL or FtsB sample, the equivalent of 7 μg or 15 μg, respectively, of total protein was separated by SDS-PAGE (Invitrogen) and transferred to polyvinylidene difluoride membrane (VWR). Horseradish peroxidase–tagged anti-FLAG (M2) antibodies (Sigma; 1:1000) were used for immunoblotting analysis.
4
Western Blot Analysis of Protein Expression
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The cells were treated similarly as in the Annexin V-PI apoptosis analysis. After 24 h of treatment, cells were lysed in radioimmunoprecipitation buffer with protease inhibitor mixture (5871, Cell Signaling Technology) by incubating for 30 min on ice. After centrifugation (14,000 × g for 15 min at 4 °C), the protein amount in the supernatant was quantified using BCA Protein Assay kit (23225, Thermo Fisher). Then, the protein was diluted with 4× LDS sample buffer (NP0007, Thermo Fisher), electrophoresed on a NuPage Novex 4–12% Bis-Tris Gel (NP0335BOX, Thermo Fisher), and transferred onto polyvinylidene difluoride membrane (29301-854, VWR International). Primary and secondary antibodies used for immunoblotting are listed in SI Appendix, Table S6 . Incubation with primary antibodies (AHR, SLC7A5) was performed at 4 °C in 5% nonfat milk (170-6404, Bio-Rad) overnight. Anti–β-actin (ACTB) was used as a loading control. The membranes were then washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Detection was performed by using Luminata Crescendo horseradish peroxidase substrate (WBLUR0500, Millipore) or SuperSignal West Femto Substrate (34096, Thermo Fisher). Immunoblot images were captured by iBright CL1500 imaging system (Thermo Fisher) and each protein was quantified using ImageJ software.
5
Western Blot Analysis of Protein Expression
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Expression level across all variants was assessed by Western blot analysis (supplementary Fig. S3). 3.0 mL of cells were pelleted and resuspended in 300 μL of lysis buffer (50 mM HEPES pH 8.0, 50 mM NaCl) with 5 mM β-Mercaptoethanol (βME). The cells were sonicated and centrifuged at 21,000 × g for 10 min before collecting the supernatant.
Total protein concentration was determined by BCA assay (Pierce). 120 μL of lysates were mixed with 40 μL of 4× LDS sample buffer (Novex, Life Technologies) with βME and boiled at 98 °C for 3 min. For each FtsL or FtsB sample, the equivalent of 7 μg or 15 μg, respectively, of total protein was separated by SDS-PAGE (Invitrogen) and transferred to polyvinylidene difluoride membrane (VWR). Horseradish peroxidase-tagged anti-FLAG (M2) antibodies (Sigma; 1:1,000) were used for immunoblotting analysis.
Total protein concentration was determined by BCA assay (Pierce). 120 μL of lysates were mixed with 40 μL of 4× LDS sample buffer (Novex, Life Technologies) with βME and boiled at 98 °C for 3 min. For each FtsL or FtsB sample, the equivalent of 7 μg or 15 μg, respectively, of total protein was separated by SDS-PAGE (Invitrogen) and transferred to polyvinylidene difluoride membrane (VWR). Horseradish peroxidase-tagged anti-FLAG (M2) antibodies (Sigma; 1:1,000) were used for immunoblotting analysis.
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