Filaments of Anabaena sp. strains from 50 ml mid-log phase cultures were collected by centrifugation, washed with 50 mM Tris-HCl buffer (pH 8.0) and sonicated in an ice-water bath by five 30 s bursts with 30 s cooling intervals. The resulting crude extracts were centrifuged at 12 000 × g for 5 min at 4°C to remove cell debris, and protein concentration was determined using the BCA™ Protein Assay kit (Thermo Fisher Scientific). For each sample, 30 μg of total protein was loaded and separated by SDS-PAGE 17% polyacrylamide gel, and transferred to a polyvinylidine fluoride membrane (Millipore). Rabbit polyclonal antibodies raised against Anabaena sp. FurA recombinant protein were used, and the blot was visualized with an Universal Hood Image Analyser (Bio-Rad).
Polyvinylidine fluoride membrane
Manufactured by Merck Group
Sourced in United States
Polyvinylidine fluoride membranes are a type of polymer-based membrane material. They are known for their chemical resistance, mechanical strength, and thermal stability. These membranes are commonly used in various laboratory and industrial applications that require filtration, separation, or purification processes.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Rabbit anti actin antibody, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ab109268, by Abcam (1 mentions)
Ab14611, by Abcam (1 mentions)
Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab2732, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Rabbit anti vamp2, by Synaptic Systems (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Anti bad, by Santa Cruz Biotechnology (1 mentions)
Cytochrome c releasing apoptosis assay kit, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
14 protocols using polyvinylidine fluoride membrane
1
Analysis of Anabaena FurA Protein
2
Western Blot Analysis of Protein Samples
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Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (sigma) containing protease inhibitor (Roche). Samples were mixed with loading buffer and heated at 95 °C for 10 min and then fractionated by SDS-PAGE. Proteins were transferred to a polyvinylidine fluoride membrane (Millipore) using Mini Trans-Blot Cell (Bio-Rad) and blocked with 1% bovine serum albumin. Blots were probed with relevant primary and secondary antibodies and proteins were detected by enhanced chemiluminescent reagent (Thermo Scientific).
3
Protein Expression Analysis of Insect Larvae
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Third instar wandering larvae were staged and single larvae were lysed in 250 μl 1% Triton X-100/PBS containing a protease inhibitor cocktail (Complete, Roche). A standard Lowry protein assay was performed and ∼10 μg of the clarified lysate was subjected to SDS-PAGE using a 10% Bis-Tris gel, and the proteins were transferred to a polyvinylidine fluoride membrane (Millipore). Tubulin antibody (1:3000) was purchased from Sigma (T9026); Mouse anti-insect cathepsin L antibody (1:4000) was purchased from R&D Systems, Inc. (MAB22591). HRP-conjugated goat anti-mouse antibodies were purchased from Millipore.
4
Quantification of LERP Protein in Larvae
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Two midguts of wild-type or LerpF6 homozygous third instar larvae were pooled and lysed in 200 μl 1% Triton X-100/PBS containing a protease inhibitor cocktail (Complete, Roche). Approximately 1/10th of the clarified lysate was subjected to SDS-PAGE using a NuPAGE 4–12% Bis-Tris gel and NuPAGE Mops SDS running buffer (Life Technologies) and the proteins were transferred to a polyvinylidine fluoride membrane (Millipore). LERP was detected with a rabbit antibody generated to a soluble form of the protein (lacking amino acids 817-886). Actin was detected with a rabbit-anti-actin antibody from Sigma (A2066).
5
Protein Expression Analysis by Western Blot
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10 μg of total protein extracted by RIPA lysis buffer (Beyotime, Shanghai, China) in each group was subjected to SDS-PAGE and subsequently transferred to polyvinylidinefluoride membrane (Millipore, Billerica, MA, USA), which was then blocked by 5% skimmed milk and incubated with the primary antibodies [anti-p-mTOR (1: 500, ab109268, Abcam), anti-mTOR (1: 1000, ab2732, Abcam), anti-PDE4B (1:1000, ab14611, Abcam) and anti-GAPDH (1:1000, ab8245, Abcam)] at 4°C overnight. Next, the membrane was incubated with the corresponding secondary antibodies (Beyotime, Shanghai, China) for 1 h at room temperature. After the membranes were washed in 1× TBST, the protein bands were developed with enhanced chemiluminescent substrate (Thermo Scientific, Carlsbad, CA, USA), and ultimately, the gray value was analyzed by Image J software (NIH-Image, Bethesda, MD, USA).
6
Protein Extraction and Analysis from Fly Heads
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Adult flies heads were collected using dual sieves (#25 and #45, Thermo, Fisher, Australia). The heads were homogenized in extraction buffer (20 mM HEPES, pH 7.0, 100 mM potassium chloride, 5% glycerol, 10 mM EDTA, 0.1% TritonX-100 and cocktail of protease inhibitors (Roche)). Protein extraction was done using the Thermo Scientific Pierce BCA protein assay protocol (#23227). Quantified lysates were eluted in SDS sample buffer and boiled for 10 min. Proteins were loaded onto 12% SDS–PAGE gels. After separation, the proteins were transferred to a polyvinylidine fluoride membrane (Merck Millipore) in transfer buffer (25 mM Tris, 0.2 M glycine and 20% v/v methanol). Sx1A was detected using Sx1A antibody (mouse anti-8c3, #AB_528484 1:1,000; Developmental Studies Hybridoma Bank). To quantify VAMP2 cleavage by TeTx/LC, third instar larvae were used for protein extraction and detected with VAMP2 antibody (rabbit anti-VAMP2, #104 202 1:1000 Synaptic Systems). Mouse anti-β-actin (#ab6276, 1:1,000 Abcam) was used to detect actin. Uncropped scans are available in Supplementary Fig. 8 .
7
Western Blot Analysis of Apoptosis Proteins
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Cells were lysed in cold radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) supplemented with protease inhibitors for 20 minutes on ice. Total proteins were isolated by centrifugation at 12,000 rpm at 4°C for 4 minutes. Protein extracts (30 µg) were subjected to sodium dodocyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidine fluoride membrane (Emd Millipore, Billerica, MA). After blocking in 5% skim milk in tris-buffered saline and 0.1% Tween 20 (TBST) for 1 hour, the membranes were probed with indicated antibodies and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The bands on the film were scanned and analyzed using Image J software. The primary antibodies were anti-Bax (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bal-2 (1:500; Santa Cruz Biotechnology), anti-Bad (1:500; Santa Cruz Biotechnology), and anti-β-actin (1:1,000; Santa Cruz Biotechnology). For Cytochrome c assays, we utilized Cytochrome c Releasing Apoptosis Assay Kit (Biovision) and followed the manufacturer's instructions. The values of protein level were normalized to the level of β-actin protein.
8
Antioxidant Enzyme Activity Assay
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All reagents and PRMI 1640 media were obtained from Sigma–Aldrich Ltd. (Beijing, China) or J&K Scientific Ltd. (Beijing, China). Antioxidant enzyme assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The BCA protein assay kit was obtained from Beyotime Institute of Biotechnology (Jiangsu, China). Polyvinylidine fluoride membranes and Centricon YM-3 were purchased from the Millipore Corp. (USA). Rabbit anti-cyt c (AF0146) and rabbit anti-VDAC1 (DF6140) were obtained from Affinity Biosciences (OH, USA). Fetal bovine serum was purchased from Hangzhou Sijiqing Biomaterials Co., Ltd. (Hangzhou, China).
1H NMR and 13C NMR spectra were recorded using a Varian Mercury spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR. HR-MS spectra were obtained on an Orbitrap Elite (Thermo Scientific) mass spectrometer. UV/Vis spectra were recorded at 25 °C with a Perkin-Elmer Lambda 25 spectrophotometer that was equipped with temperature-controlled cell holders (USA). Fluorescence intensity and fluorescence spectra were measured at 25 °C with an Infinite M200 Pro Multimode Reader (Switzerland) and a Shimadzu RF-5301 spectrofluorimeter (Japan), respectively.
1H NMR and 13C NMR spectra were recorded using a Varian Mercury spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR. HR-MS spectra were obtained on an Orbitrap Elite (Thermo Scientific) mass spectrometer. UV/Vis spectra were recorded at 25 °C with a Perkin-Elmer Lambda 25 spectrophotometer that was equipped with temperature-controlled cell holders (USA). Fluorescence intensity and fluorescence spectra were measured at 25 °C with an Infinite M200 Pro Multimode Reader (Switzerland) and a Shimadzu RF-5301 spectrofluorimeter (Japan), respectively.
9
Western Blot Analysis of Cellular Proteins
10
Protein Extraction and Western Blot Analysis
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Protein extraction from dissected tissues was performed as previously described [70 (link)]. Extracts were separated by electrophoresis in a 10% SDS-polyacrylamide gel at constant current of 120 volt. Proteins were transferred to polyvinylidinefluoride membranes (Millipore Corporation, Billerica, MA, USA), subsequently blocked 5% dry milk in TBST (Tris Buffered Saline with 0.1% Tween 20) for 1 h at room temperature and then incubated with anti-Dfr S/L, anti-Phm, anti-Dib, or anti-Actin (mAbcam 8224) as primary antibodies, and with ECLTM anti-rat IgG (Amersham), ECLTM anti-mouse IgG (GE Healthcare), and ECLTM anti-rabbit IgG (GE Healthcare) as 2nd antibodies. The blot was developed using either SuperSignalTM West Femto maximum sensitivity substrate or SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) according to the manufacturers’ instructions. Digital images were acquired with ChemiDoc™ Imaging Systems (Bio-Rad). Protein levels were quantified with Image Lab™ Software (Bio-Rad) and normalized against Actin or Lamin. Statistics was performed using two-way ANOVA.
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