Blood from an infected mouse was diluted in ookinete culture medium and incubated at 19°C for 8 h. Half of the culture was then pelleted (retort sample) and red blood cells lysed with ammonium chloride. After another 16 h ookinetes were enriched in the same way. The two samples were processed similarly and in parallel. They were lysed by sonication in the presence of a cocktail of protease inhibitors (Sigma P8340) and 100 μΜ PMSF. The samples were then pelleted at 700xg for 10 min to remove unbroken cells. The samples were then centrifuged at 15 000xg for 10 min and the supernatant were processed for SDS-PAGE and analyzed by Western blot. The blots were probed with the monoclonal anti-mCherry antibody. The secondary anti-rabbit antibody was conjugated with horse radish peroxide. The signal was detected using the SuperSignal West Pico solution (Pierce Biotechnology).
Supersignal west pico solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal West Pico Solutions is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. The solution contains a stable peroxide solution and an enhanced luminol solution, which when combined, produce a luminescent signal that can be detected using imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Igg hrp conjugated peroxidase secondary antibodies, by Cell Signaling Technology (2 mentions)
G box hr16 imaging system, by Syngene (2 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (1 mentions)
Horseradish peroxidase coupled goat anti rabbit secondary antiserum, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Nupage lds buffer, by Thermo Fisher Scientific (1 mentions)
Las 3000 system, by Fujifilm (1 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
Anti cdk1, by Santa Cruz Biotechnology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti γ tubulin, by Cell Signaling Technology (1 mentions)
Rpl13a, by Cell Signaling Technology (1 mentions)
Anti nucleolin, by Santa Cruz Biotechnology (1 mentions)
Anti γh2ax, by Abcam (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Hrp conjugated igg secondary antibody, by Cell Signaling Technology (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
13 protocols using supersignal west pico solution
1
Ookinete Enrichment and Western Blot
2
Western Blot Protein Detection
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Parasites were lysed by sonication, and the crude lysates were loaded on a denaturing 12% SDS-PAGE gel. After gel electrophoresis the proteins were transferred to nitrocellulose membrane filters by electroblotting. The membrane filters were incubated with the primary antibodies, followed by secondary antibodies conjugated with horse radish peroxide. The signal was detected using the SuperSignal West Pico solution (Pierce Biotechnology).
3
Immunoblot Analysis of PEN2 Protein
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Whole rosette leaves of 4-week-old plants were harvested at the indicated time points after powdery mildew inoculation, frozen in liquid nitrogen and homogenized. Total protein extracts were prepared in 120 μl/100 mg fresh weight (FW) lysis buffer (20 mM HEPES pH 7.5; 13% sucrose; 1 mM EDTA; 1 mM DTT; 0.01% Triton X-100, complete Mini protease inhibitor cocktail tablet/10 ml buffer (Roche). Cell debris was removed by centrifugation, the protein concentration was determined by Bradford assay and protein samples were boiled for 10 min in 2x loading buffer. Following gel electrophoresis, proteins were blotted onto nitrocellulose membranes and the transfer visualized with the Ponceau protein dye. Blots were incubated with PEN2 antiserum (1:5,000 dilution in PBS-T at 4°C overnight; Lipka et al., 2005 (link)) and subsequently with a horseradish peroxidase-coupled goat anti rabbit secondary antiserum (Santa Cruz Biotechnology, Dallas, USA; 1:5000 dilution in PBS-T). For signal detection, the SuperSignal West Pico solution (Thermo Fisher Scientific, Waltham, USA) was used according to the manufacturer's instructions. Luminescence was documented on X-ray films.
4
Immunoblotting of CAR-Transduced T Cells
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CAR transduced Jurkat, KHYG-1, and T cells were harvested and cell lysates were made by 1× sample buffer (10% glycerol, 2% SDS, 50 mM Tris-HCl (pH 6.8), 3% β-mercaptoethanol). Lysates were boiled 10 min, at 95 °C. Samples were loaded on 4–15% gradient Mini-PROTEAN TGX gels (456-1086; Bio-Rad Laboratories) and electrophoresis was performed at 100 V for 75 min. The transfer step was conducted at 250 mA for 60 min. Using the blocking buffer (5% skimmed milk in 1× TBS-T), membrane was blocked for 60 min. After washing with 1X TBS-T, the membrane was incubated with primary antibody (CD3ζ (551034; BD Biosciences, San Jose, CA, USA), GAPDH (5174; Cell Signaling Technology, Danvers, MA, USA)) overnight at 4 °C. Next day, the membrane was washed with 1× TBS-T and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (31430 and 31460; Thermo Fisher Scientific). Signal intensity was detected using SuperSignal West Pico solution (1863096 and 1863097; Thermo Fisher Scientific) and Sensi-Q2000 Chemidoc (LugenSci, Bucheon, Korea) instruments.
5
Protein Extraction and Western Blotting
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Cells were adjusted to equal numbers, washed with PBS, resuspended in triton lysis buffer (20 mM Tris–HCl, 150 M NaCl, 1% Triton X‐100, 1 mM EDTA, 1 mM EGTA, 1 mM b‐glycerolphosphate, 2.5 mM sodium pyrophosphate and 1 mM sodium orthovanadate), incubated for 30min on ice and then spun down at full speed for 7 min to clear lysates. For brain tissue samples, forebrains were dissected, resuspended in triton lysis buffer and sonicated in a water bath for 15 min before being processing as described above. Lysates were heated at 70°C in NuPage LDS buffer (Thermo Fisher, NP0007) with 50 mM DTT. Samples were separated on 7.5% polyacrylamide gels, transferred to nitrocellulose and blocked with 5% skim‐milk powder or 5% BSA in Tris‐buffered saline with 0.05% Tween‐20 (TBST) for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. After 3 washes in TBST, membranes were incubated with peroxidase‐linked secondary antibodies for 1h at RT. Following an additional 3 washes, membranes were treated with Supersignal West Pico solution (Thermo Scientific, 34579). Images were acquired on an LAS‐3000 system (Fuji Film).
6
Whole Cell Extract Preparation and Western Blot
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Whole cell extracts were prepared by resuspending the pellets in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8) containing protease inhibitors (100 μM PMSF, 1 mM Benzamidine, 2.5 μg/ml Pepstatin A, 10 μg/ml Leupeptin, and 10 μg/ml Aprotinin) and phosphatase inhibitors (1 mM each of NaF, Na3VO4, and Na2P2O7). Protein concentration was measured using the BCA protein assay kit (Pierce, #23227). Equal amounts of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Millipore, HATF304F0). Transfer efficiency and equal loading were confirmed by Ponceau S staining. Membranes were blocked overnight at 4°C with 5% nonfat milk in TBS-T, followed by incubation in primary and secondary antibodies (1 hour at RT, 2% milk in TBS-T). Protein bands were visualized using SuperSignal West Pico solutions (Thermo Scientific).
7
Comprehensive Protein Analysis Pipeline
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Protein isolation, SDS PAGE, and Western blotting were performed as described in Nagy et al. [102 (link)]. Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% TritonX 100, 0.5% sodium deoxycholate, 1mM EDTA, 1mM Na3VO4, 1 mM PSMF, 1 mM NaF, and protease inhibitor cocktail). Protein extracts (20–50 μg) were separated on 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes by electroblotting. After blocking for 1 h in TBST containing 5% BSA, the membranes were incubated with primary antibodies overnight at 4 °C. The membranes were washed with 1× TBST solution, then probed with IgG HRP-conjugated peroxidase secondary antibodies (1:2000, Cell Signaling Technology, Inc, Beverly, MA, USA). Bands were visualized by enhanced chemiluminescence (SuperSignal West Pico Solutions, Thermo Fisher Scientific Inc., Rockford, IL, USA). Blots were quantified by densitometry using the Image J software and the results of densitometry is uploaded alongside with the primary data to Figshare.com (https://figshare.com/s/81c2f5906706c60e6c3f ). The primary and secondary antibodies are listed in Table 5 .
8
SDS-PAGE and Western Blotting Protocol
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SDS PAGE and Western blotting was performed as in [60 (link)]. Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% TritonX 100, 0.5% sodium deoxycolate, 1 mM EDTA, 1 mM Na3VO4, 1 mM PSMF, 1 mM NaF, protease inhibitor cocktail). Protein extracts (20–50 μg) were separated on 10% SDS polyacrylamide gels and blotted onto nitrocellulose membranes. Then, membranes were blocked in TBST containing 5% BSA for 1 h and incubated with primary antibodies overnight at 4 °C. After washing with 1 × TBST solution, the membranes were probed with IgG HRP-conjugated peroxidase secondary antibodies (1:2000, Cell Signaling Technology, Inc., Beverly, MA, USA). Bands were visualized by enhanced chemiluminescence reaction (SuperSignal West Pico Solutions, Thermo Fisher Scientific Inc., Rockford, IL, USA). Blots were quantified by densitometry using the Image J software. Densitometric analysis is represented as a separate supplementary file, and it is also uploaded among the primary data. The primary and secondary antibodies are listed in Table 3 .
9
Western Blotting of Mouse Muscle Proteins
10
Western Blot Analysis of Cellular Proteins
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