Total protein was extracted using 2X SDS Lysis Buffer (100 mM Tris, 4% SDS, 10% glycerol, 200 mM NaCl, 2 mM EDTA, pH=6.8) from SW1116 and HCT116 cells, and then 30 g protein was separated by SDS-PAGE with 10% acrylamide and transferred onto polyvinylidene fluoride membranes. The membranes were probed with specific antibodies against STIM1 (cat. no. sc-166840; 1:1,000 for HCT116 cells and 1:500 for SW1116 cells; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), poly(ADP-ribose) polymerase (PARP; cat. no. 9542; 1:1,000; Cell Signaling Technology Europe, B.V., Leiden, The Netherlands) or GAPDH (cat. no. 10494-1-AP; 1:50,000 for HCT116 cells and 1:10,000 for SW1116 cells; Proteintech Group Inc., Chicago, IL, USA) at 4°C overnight. Membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. sc-2005; 1:5,000; Santa Cruz Biotechnology, Inc.) or goat anti-rabbit (cat. no. sc-2054, 1:5,000; Santa Cruz Biotechnology, Inc.) immunoglobulin G antibody and visualized using the Pierce enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.
Pierce enhanced chemiluminescence kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce enhanced chemiluminescence kit is a laboratory reagent used for the detection and quantification of proteins in Western blot analysis. The kit contains the necessary components to generate a chemiluminescent signal when the target protein is bound by a specific antibody, allowing for visualization and analysis of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Poly adp ribose polymerase parp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit, by Abcam (1 mentions)
Anti β actin rabbit monoclonal antibody, by Abcam (1 mentions)
2 protocols using pierce enhanced chemiluminescence kit
1
Quantification of STIM1 and PARP Protein Expression
2
Quantitative Western Blot Analysis
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Protein samples were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer’s protocols. Protein samples were heated with SDS sample buffer (0.25 M Tris–HCl, pH 6.8, 0.5 M DTT, 10% SDS, 50% glycerol and 0.5% bromophenol blue) at 95 °C for 5 min, a total of 30 μg were loaded per lane and then separated on 15% SDS-PAGE and electroblotted onto polyvinylidene fluoride membranes (GE10600023, GE Healthcare Life Sciences, Logan, UT, USA). Subsequently, the membranes were incubated with one of the primary rabbit polyclonal antibodies at a dilution of 1:1,000 (Abcam) or anti-β-actin rabbit monoclonal antibody at a dilution of 1:5,000 (Abcam) overnight at 4 ℃. Immunoreactive bands were detected by incubation with horseradish peroxidase-conjugated goat anti-rabbit (Abcam) at a dilution of 1:5,000 for 2 h at room temperature. Detection by chemiluminescence was performed using a Pierce enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
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