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Thermo scientific supersignal west pico chemiluminescent substrate

Manufactured by Thermo Fisher Scientific
Sourced in United States

The Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate is a laboratory product designed to detect and quantify proteins in Western blot analysis. It is a sensitive chemiluminescent detection reagent that produces a luminescent signal when it interacts with the target protein. The substrate is suitable for use with a variety of detection systems, including X-ray film and digital imaging systems.

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7 protocols using thermo scientific supersignal west pico chemiluminescent substrate

1

Western Blot Profiling of Cellular Proteins

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Whole-cell extracts were lysed in RIPA buffer (SigmaAldrich, Waltham, MA, USA) supplemented with 1% Halt Protease Inhibitor. Protein was quantified using the Bradford reaction. 10μg of each sample was loaded into individual lanes and subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight in primary antibodies against BTG-3 (1:1000) (Abcam, UK), HECW2 (1:1000) (SigmaAldrich, USA), and actin (1:3000) (Cell Signaling, USA) at 4 °C in 5% milk in 1% tween in tris buffered saline. Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG or anti-rabbit IgG secondary antibodies (BioRad) were used, and the signal was detected using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA).
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2

Western Blot Protein Analysis Protocol

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Cells were harvested after incubation periods as indicated, washed with PBS, and lysed in lysis buffer (20 mM HEPES, pH 7.9; 350 mM NaCl; 1 mM MgCl2; 0.5 mM EDTA, pH 8.0; 0.1 mM EGTA, pH 8.0) supplemented with protease and phosphatase inhibitors for 20 min on ice. Protein concentrations were quantified by Bradford assay. 4× loading buffer (200 mM Tris, pH 6.8; 40% glycerol; 8% SDS; 4% β-mercaptoethanol; 50 mM EDTA, pH 8.0; 0.01% bromophenol blue) was added and the lysates were boiled to 96 °C for 5 min. After separation by dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were blotted on nitrocellulose membranes and visualized by Ponceau S (Sigma-Aldrich, St. Louis, MO, USA) staining. Before incubation with primary and HRP-conjugated secondary antibodies, the membranes were blocked in Tris-buffered saline with 5% milk powder and 0.1% Tween 20. The protein bands were detected by Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) and then visualized on Amersham Hyperfilm ECL (GE Healthcare, Freiburg, Germany). The area of protein bands determined by Image J software was used to perform normalization to β-actin and to calculate relative expression subsequently [59 (link)].
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3

Amyloid-Beta Protein Analysis by Western Blot

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The samples from the inhibition and disaggregation experiments were analyzed by gel electrophoresis with Western blot using an anti-Aβ antibody (6E10)20 (link)28 (link). Each sample (10 μL) was separated on a 10–20% Tris-tricine gel (Invitrogen, Grand Island, NY, USA). Following separation, the gel was transferred onto nitrocellulose membrane which was blocked with bovine serum albumin (BSA, 3% w/v, Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 3 h at room temperature. The membrane was treated with antibody (6E10, Covance, Princeton, NJ, USA; 1:2000) in a solution of BSA (2% w/v) in TBS-T overnight at 4 °C. Following washing, the membrane was treated with horseradish peroxidase-conjugated goat antimouse secondary antibody (1:5000; Cayman Chemical, Ann Arbor, MI, USA) in 2% BSA in TBS-T solution for 1 h at room temperature. Protein bands were visualized using ThermoScientific Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
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4

Immunoblot Analysis of GlpQ Antibodies

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Purified GlpQ (500 ng) was electrophoresed on each replicate lane of a precast mini 4%�?"20% sodium dodecyl sulfate�?"polyacrylamide gel electrophoresis gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a nitrocellulose membrane using the Bio-Rad MiniTrans Blot Cell (Bio-Rad Laboratories). Replicate strips containing rGlpQ were blocked overnight at 4A�C in PBS (pH 7.2)/5% dried milk/0.05% Tween 20. The blocked strips were then individually incubated with human serum at a 1:250 dilution at room temperature in PBS (pH 7.2)/2.5% dried milk/0.05% Tween 20 for 1 h. The strips were then washed 3 times and incubated for 1 h with horseradish peroxidase�?"conjugated rabbit anti-human IgG (Sigma-Aldrich, St. Louis, MO, USA) or with horseradish peroxidase�?"conjugated goat anti-human IgM (Invitrogen) at a 1:5,000 dilution in PBS (pH 7.2)/2.5% dried milk/0.05% Tween 20. Bound antibodies were detected by using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Serum from �%^10% of the study participants reacted to a �%^55-kDa band, presumably a trace contaminant copurified with the rGlpQ generated in a bacterial expression system. Samples with a 39-kDa band corresponding to GlpQ on positive control mouse serum samples were considered GlpQ antibody�?"positive (Figure 1).
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5

Western Blot Analysis of JAK Proteins

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Protein lysates were prepared in RIPA buffer (20mM Tris-HCl pH 7.4, 150mM NaCl, 1% Triton-X-100, 0.1% SDS, 0.5% sodium deoxycholate). Western blot analysis was performed on HUT78 cell lysates separated by SDS-PAGE and transferred to nitrocellulose membranes using standard procedures. For immunoblot detection, we used the following antibodies: JAK1 (dilution 1:1000) (Cell Signaling Technology, Cat. No. 3332), JAK2 (dilution 1:1/500) (Santa Cruz Biotechnology , Cat. No. sc390539) or GAPDH (D16H11) (dilution 1:1000)(Cell Signaling Technology, Cat. No. 5174). The blots were developed with Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Cat. No. 34080) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Cat. No. 34094) according to the manufacturer’s instructions.
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6

Quantifying GFAP, ICAM-1, and Caspase-3 in Rat Brain

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Western blot analysis was conducted following previous reports.36 (link)–38 (link) The rat brain tissue was homogenized in protein lysate buffer. The homogenate was subjected to 10% SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane. After blocking with 5% skim milk in TBST for 1 h, the membrane was incubated with primary antibody (against active GFAP, ICAM-1, and caspase-3) at 4°C overnight and subsequently treated with alkaline phosphatase-conjugated secondary antibodies. GFAP, ICAM-1, and caspase-3 were developed using BCIP/NBT. Blots were stained with anti-β-actin antibody, and the quantification of proteins was normalized based on β-actin band density. The antigen–antibody products were detected with Thermo Scientific Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA). The results were analyzed with a FluorChem™ system (Alpha Innotech, San Leandro, CA, USA).
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7

Immunoblotting of IL-18 Protein Expression

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Proteins from each group of cells were isolated 24 h and 96 h after lentiviral expression vector transduction. The protein concentration was determined using the bicinchoninic acid protein assay. After adjusting the protein concentration of each sample, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide gel) and transferred to a polyvinylidene fluoride membrane following electrophoresis. The membrane was blocked at 4°C with 5% nonfat dry milk for 2 h and then incubated overnight at 4°C on a rocking platform with IL-18 (1:1000) or internal reference (β-actin, 1:1000) primary antibodies. Next, the membrane was washed with Tris-Buffered Saline and Tween (TBST) and incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibodies (1:5000). Finally, the membrane was treated with Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and exposed to films to detect the protein bands.
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