Protein concentration was measured by staining with amido black assay (Merck) according to the manufacturer's specifications. Per sample, 50 μg of proteins and 5 μl of PageRuler Prestained Protein Ladder (SM0671, Fermentas) were fractionated by electrophoresis through 12% SDS-polyacrylamide gels (SDS-PAGE) and transferred to Protran nitrocellulose membranes (Whatman) by semi-dry blotting using Tris-base, Glycerol, (Merck), 10 % SDS, methanol (J.T.Baker, The Netherlands) in double-distilled water for 30 min with a constant current at 100 mA per membrane. The membranes were blocked with 5% non-fat dry milk (AppliChem GmbH) in TBST (Tris-buffered saline (TBS) + 0.05% Tween®-20, Serva) or with 3% BSA in TBST (Sigma-Aldrich, München). For the detection of active RAL and RAS proteins, we used the following primary mouse monoclonal antibodies: RALA included in STA-408-CB and pan-RAS included in STA-400-CB pull-down assays, RALB: #3523, KRAS: #2146 and beta-tubulin: #TA801672 (Cell Signaling). Anti-mouse IgG-Horseradish Peroxidase (HRP)-conjugate (1:5000 dilution, 325-035-045, Dianova) or anti-rabbit IgG-HRP-conjugate (1:10, 000 dilution; 7074, Cell Signaling) were used as secondary antibody. Protein bands were visualized using the ECL chemoluminescence detection system (GE Health Care) on X-ray films (Amersham Hyperfilm e).
Protran nitrocellulose membrane
Manufactured by Cytiva
Sourced in Germany, United Kingdom, United States
The Protran nitrocellulose membrane is a porous, high-binding support material commonly used in various biological and analytical techniques. It is designed to provide efficient and reliable protein transfer and immobilization for applications such as Western blotting and dot blotting.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Boster Bio (4 mentions)
Amersham ecl kit, by Cytiva (4 mentions)
Protein assay, by Bio-Rad (3 mentions)
Nupage 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Mops buffer, by Thermo Fisher Scientific (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Dot blot apparatus, by GE Healthcare (3 mentions)
Globulin free bsa, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Sheep anti mouse igg hrp, by Cytiva (2 mentions)
Gel doc system, by Uvitec (2 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (2 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (2 mentions)
Xcell surelock electrophoresis system, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
84 protocols using protran nitrocellulose membrane
1
Protein Concentration Measurement and Western Blot Analysis
2
Western Blot Analysis of Ovarian Markers
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Western blot analysis for LATS2, YAP1, FSHR, STAR, CYP11A1, ESR1, and ESR2 was performed using total cellular extracts as previously described (Xu et al., 2018a (link),b (link)). Briefly, equal amounts of protein were separated by 10% (w/v) SDS-polyacrylamide gel under reducing conditions and electrotransferred to Protran nitrocellulose membranes (Whatman, Dassel, Germany). After electrophoresis of the protein samples in a mini gel apparatus, a prestained protein molecular weight marker was loaded to locate/monitor the target proteins in the electrophoresis (SDS-PAGE). At the approximated protein size position, the gel was directly cut and transferred to the nitrocellulose membrane for western blotting. The affinity-purified antibodies for LATS2 and the other antibodies were used (Supplementary Table S3 ). The horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody was incubated for 2 h at room temperature. Blots were subsequently performed with ECL western blotting agent (Rockford, IL) for 5 min and exposed to X-ray film for 1 to 5 min. The signals were detected using the ECL Plus Western blotting detection system according to the manufacturer's instructions. Anti-β-actin (dilution 1:1,000, Boster, Wuhan, China) antibody acted as a loading control. As a result, the molecular size of the ladders was not observed in the original blots.
3
Western Blot Analysis of SERT Protein
4
Western Blot Analysis of Autotaxin in Plasma
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1 μl of plasma samples were diluted in Laemmli buffer, electrophorised on 8% SDS-polyacrylamide gels and transferred to Protran nitrocellulose membranes (Whatman plc, Maidstone, Kent, UK) using the Trans-Blot SD Semi-Dry Transfer system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Primary a-ATX Ab incubation (rat monoclonal 4F1, kindly provided by J. Aoki, 1:1000; rabbit polyclonal Cayman 1:250) was performed overnight in 5% (w/v) non-fat milk in TBS-Tween 0.05% (TBST) at 4°C. The membranes were then washed three times with TBST and incubated with HRP-conjugated secondary Abs (a-rat or a-rabbit respectively; 1:2000) for one hour at room temperature. Membranes were washed three times with TBST and antibody-antigen complexes were revealed using ECL chemiluminescent reagent (Pierce/Thermo Scientific, Rockford, IL, USA). Antibody specificity has been scrutinized previously [13 (link)].
Plasma biochemical and haematological analyses were performed with the Abbott Architect 8200 and Cell-Dyn 3700 analysers respectively.
Plasma biochemical and haematological analyses were performed with the Abbott Architect 8200 and Cell-Dyn 3700 analysers respectively.
5
Impact of Conditional Overexpression on Seed Germination
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To analyze the impact of the conditional overexpression on seed germination, 50 seeds of each tested genotype were sown on Protran® nitrocellulose membranes (Whatman) on MS medium supplemented with 10 μM ß-estradiol. The seeds were kept in a growth chamber under previously described standard conditions. The germination process of the different genotypes was periodically monitored under a Leica MZ10F stereomicroscope. To ensure equal conditions, all seeds were harvested at the same time, when the entire siliques had browned. The experiment was carried out in three biological replicates.
6
Extracting and Analyzing Total Protein
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For total protein extraction, equal amounts of 7-day-old seedlings were grounded into fine powder as described above, and then resuspended in 2× SDS buffer. The samples were lysed on ice for 30 min, then centrifuged at 18,514 g for 10 min at 4°C. The supernatants were transferred into new tubes, and heated in boiling water for 10 min. 10 μL of total protein extracts were separated via 10% SDS-PAGE and then transferred to Protran nitrocellulose membranes (Whatman). The immunoblots were performed using an anti-BIN2 antibody (Agrisera; cat. no. AS163203; 1:5,000 dilution) in Supplemental Figure S2B , and an anti-GFP antibody (Abways; cat. no. AB0005; 1:5,000 dilution) and an anti-Tubulin antibody (Abmart, cat. no. M20045; 1:5,000 dilution) in Figure 5, A and H at 4°C overnight. The secondary antibodies used were Goat anti-mouse lgG (H + L) HRP (ShareBio; SB-AB0102; 1:5,000) or Goat anti-Rabbit lgG (H + L) HRP (ShareBio; SB-AB0101; 1:5,000). All antibodies were diluted in 5% skim milk powder (in PBS containing 1% Tween-20). The images were taken using the BIO-RAD ChemiDoc Imaging System. The integrated optical density (IOD) values of VLG and tubulin protein band signals in Figure 5, A and H were quantified using Image Lab software (Bio-Rad) (Ye et al., 2019 (link)).
7
Characterization of Cell Signaling Pathways
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Following the cell transfection, a western blot analysis of SLIT2, ROBO1/2, RAFs, CDC42 and RAC1 proteins and phosphorylated PAKs, RAFs, MEK1/2 and ERK1/2 kinases was conducted as previously described3 (link) using total cellular extracts. Briefly, equivalent amounts of protein were separated by 10% (w/v) SDS-polyacrylamide gel under reducing conditions and electro-transferred to Protran nitrocellulose membranes (Whatman, Dassel, Germany). Affinity-purified antibodies against SLIT2 and the other proteins were used (Table S3 ). The samples were incubated with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody for 2 h at room temperature. The blots were subsequently incubated with the ECL western blotting reagent (Rockford, IL, USA) for 5 min and exposed to X-ray film for 1–5 min. The outcome was visualized by an ECL Plus western blotting detection system according to the manufacturer’s instructions. An anti-β-actin antibody (dilution 1:1000, Boster, Wuhan, China) was used as a loading control.
8
Western Blotting Protocol for Protein Detection
9
Western Blot Protein Analysis Protocol
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Cells were directly lysed with Laemmli sample buffer containing protease inhibitors (Roche). Cell lysates were loaded onto NuPage 12% Bis-Tris gels (Life technologies) and separated by electrophoresis in MOPS buffer (Life technologies). Proteins were transferred onto Protran nitrocellulose membranes (Whatman) and saturated for 1 h in PBS-T (PBS, 0.1% Tween 20) supplemented with 5% non-fatty milk. Incubation with primary antibodies was performed overnight at 4°C. The membranes were then washed three times with PBS-T and incubated with appropriate secondary antibodies for 1 h. Membranes were washed three times with PBS and proteins revealed either with an Odyssey imaging system (LI-COR Bioscience) or with an Uvitec gel doc system (Uvitec Cambridge).
10
Western Blot Analysis of Transcription Factors
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Following cell transfection test, western blot analysis for FOXL2, phospho-Smad1, 2 and3 and total Smad1, 2 and 3 protein was conducted as previously described [35 (link)] using total cellular extracts. Briefly, equivalent amounts of protein were separated by 10% (w/v) SDS- polyacrylamide gel under reducing conditions and electro-transferred to Protran nitrocellulose membranes (Whatman, Dassel, Germany). The affinity purified antibody for FOXL2 (Santa Cruz, CA, USA) and (phospho-) Smad1 (Rockford, IL, USA), Smad 2 and3 (Invitrogen, Carlsbad, CA, USA) was used (S2 Table ). The horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody was incubated for 2 h at room temperature. Blots were subsequently performed with ECL western blotting agent (Rockford, IL, USA) for 5 min and exposed to X-ray film for 1–5 min. The outcome was visualized by the ECL Plus Western blotting detection system according to the manufacturer's instructions. Anti-β-actin (dilution 1:1000, Boster, China) antibody acted as loading control.
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