Concentrated supernatants obtained for the hemolysis assay were separated electrophoretically using a 5% stacking and a 10% running sodium dodecyl sulfate-polyacrylamide gel and were transferred to a polyvinylidene fluoride membrane (Merck). The membranes were blocked for 1 h at room temperature (RT) with 5% skimmed milk powder in Tris-buffered saline (TBS) with 1% Tween®20 (Carl Roth). Afterward, they were incubated with a polyclonal rabbit antiserum raised against Ply (Davids Biotechnologie GmbH, Germany; dilution 1:500 in 1% skimmed milk powder in TBS with 1% Tween®20) [38 (link)] overnight at 4°C, followed by incubation with HRP-linked goat anti-rabbit IgG (Cell Signaling; dilution 1:5,000 in 1% skimmed milk powder in TBS with 1% Tween®20) for 1 h at RT. The membranes were developed with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific), and the chemiluminescent signal was detected with ChemoCam Imager 3.2 (Intas, Germany).
Tween 20
Manufactured by Cell Signaling Technology
Sourced in Germany
Tween 20 is a non-ionic surfactant commonly used in various laboratory protocols. It is a polyoxyethylene derivative of sorbitan monolaurate, which acts as a detergent to solubilize and stabilize proteins. Tween 20 is widely used in biochemical and immunological techniques, such as Western blotting, ELISA, and protein purification, to prevent non-specific binding and improve the efficiency of these assays.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate enhanced chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Anti human igg hrp, by Southern Biotech (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Tris buffered saline (tbs), by Cell Signaling Technology (2 mentions)
Tween 20, by Carl Roth (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Chemocam imager 3, by Intas (1 mentions)
Hrp linked goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Tween 20, by Merck Group (1 mentions)
Anti phospho smad2 ser465 467 138d4, by Cell Signaling Technology (1 mentions)
Lumi lightplus western blotting substrate, by Roche (1 mentions)
Cleaved caspase 3 9661, by Cell Signaling Technology (1 mentions)
Ps6 4858, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
7 protocols using tween 20
1
Western Blot Analysis of Pneumolysin
2
Peptide Microarray Immunoassay Protocol
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Nitrocellulose membrane was marked with pencil to indicate the regions where the peptides would be spotted. Four micrograms of recombinant peptide was spotted onto each marked region of the nitrocellulose membrane, then the membrane was cut into strips and blocked in Tris Buffered Saline with Tween 20 (Cell Signaling Technology) and 5% bovine serum albumin (TBST 5% BSA) for 30 min at room temperature. The membrane was next incubated in cell culture supernatant from recombinant antibody expression diluted 1:20 in TBST 5% BSA for 1 hour at room temperature. Nitrocellulose was washed three times with TBST for 5 min per wash, then incubated with anti-human IgG-HRP (Southern Biotech) diluted 1:500 in TBST 5% BSA for 1 hour at room temperature. Nitrocellulose was washed three times with TBST for 5min per wash, then once with TBS for 5 min. Nitrocellulose was incubated with SuperSignal West Femto Maximum Sensitivity Substrate enhanced chemiluminescent substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions and chemiluminescent signals were captured using a CCD camera-based imager.
3
Protein Lysate Preparation and Western Blot
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To prepare protein lysates, matrigel was mechanically disrupted and resuspended in PBS. Organoids were pelleted and washed once with PBS. Organoids were resuspended in 1X reducing sample buffer, and lysates were sonicated and boiled for 5 min at 85°C. Protein concentration was determined using the Protein Quantification Assay from Macherey‐Nagel. Ten micrograms of protein was loaded on 4–15% Mini‐PROTEAN® TGX™ Precast Protein Gels (Bio‐Rad) and transferred to a hydrophobic, microporous, polyvinylidene difluoride (PVDF) membrane with a pore size of 0.2 μm (Roche). Membranes were blocked for 1 h in 5% bovine serum albumin in PBS + 0.2% Tween‐20 (Sigma) and subsequently incubated in primary antibody dilution in 5% bovine serum albumin in PBS + 0.2% Tween‐20 at 4°C overnight [anti‐phospho‐Smad2 (Ser465/467) (138D4) (1:2,000, Cell Signaling 3108); anti‐Smad2 (L16D3) (1:2,000, Cell Signaling 3103)]. The secondary antibody in 5% bovine serum albumin in PBS + 0.2% Tween‐20 [anti‐rabbit‐HRP (1:2,000 Cell Signaling 7074); anti‐mouse‐HRP (1:10,000, Southern Biotech 1070‐05)] was applied for 1 h at room temperature. After washing, the membrane was developed using Lumi‐LightPLUS Western Blotting Substrate (Roche), and signal was detected with the ImageQuant LAS4000 (GE Healthcare Life Sciences). Washing steps were performed using PBS + 0.2% Tween‐20.
4
Western Blot Analysis of Apoptosis Markers
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SKLMS-1- and SW982-treated cells were lysed with radioimmunoprecipitation assay buffer containing protease inhibitors (1 mmol l−1 phenylmethylsulfonyl fluoride, 10 mg ml−1 aprotinin, and 10 mg ml−1 leupeptin) and the lysates were centrifuged at 13 000 × g, at 4°C, for 30 min. Lysate aliquots (50 μg) were resolved by 10% SDS–PAGE and transferred onto nitrocellulose membranes. After blocking with 5% skimmed milk in PBS containing 0.2% Tween 20 (Dallas, TX, USA) at room temperature for 1 h, membranes were incubated overnight at 4°C with the appropriate primary antibody (cleaved caspase 3 #9661, native S6 #2217, and pS6 #4858 from Cell Signaling Technology). Blots were then incubated at room temperature for 1 h with a horseradish peroxidase-conjugated secondary antibody and the peroxidase activity was detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA) following the instructions of the manufacturer. Immunodetection of α-tubulin was used as a loading reference.
5
Western Blot Analysis of Endophilin A2
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Whole cell lysates from DA and DAMut rats were obtained from brain samples by lysis in RIPA buffer (ThermoScientific). Samples were run in a NuPAGE 4–12% Bis-Tris gel (Novex, Invitrogen) and transferred to a PVDF membrane (EMD, Millipore). Blots were blocked with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween 20 (Cell Signaling Technology) and subsequently incubated with mouse anti-rat Endophilin A2 (clone S51-1, Origene Technologies, Inc) and rabbit anti-histone 2B (Cat. No C49810, LifeSpan Biosciences, Inc). Blot was incubated with peroxidase-conjugated goat anti-mouse IgG(Cat no. 115-035-062, Jackson ImmunoResearch laboratories Inc.) or peroxidase-conjugated donkey anti-rabbit IgG (Cat no.771-036-152, Jackson ImmunoResearch laboratories Inc.) respectively and developed using enhanced chemiluminescence. Pictures including the entire blot can be found in the Source Data file.
6
Western Blot Analysis of Signaling Proteins
7
Peptide Microarray Immunoassay Protocol
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