Human U251 and T98G glioblastoma cells were lysed in RIPA buffer (50 mM Tris-HCl, Invitrogen). The extracted proteins were electroseparated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes (Amersham Biosciences). The primary antibody against human FGFR3 (1:200, Santa Cruz Biotechnology) was applied overnight at 4°C. Horseradish peroxidase-linked secondary antibody was applied for 2 hours at room temperature. Glyceraldehyde-3-phosphate dehydrogenase was used as the internal control and blots were visualized by an enhanced chemiluminesence system (Amersham Biosciences) as per the manufacturer’s protocol.
Enhanced chemiluminesence system
Manufactured by Cytiva
Sourced in United States
The Enhanced Chemiluminescence System is a laboratory equipment designed to detect and quantify proteins in Western blot analysis. It utilizes chemiluminescent substrates to generate light signals proportional to the amount of target proteins, which are then captured and analyzed using specialized imaging hardware and software.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Igf1r, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phospho erk1 2 p erk1 2, by Cell Signaling Technology (1 mentions)
Phosphor akt p akt, by Cell Signaling Technology (1 mentions)
Thy 1, by Santa Cruz Biotechnology (1 mentions)
Phosphorylated trkb, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Tris based lysis buffer, by Thermo Fisher Scientific (1 mentions)
β actin primary antibody, by Abcam (1 mentions)
Sds page gel, by Thermo Fisher Scientific (1 mentions)
3 protocols using enhanced chemiluminesence system
1
Western Blot Analysis of FGFR3 in Glioblastoma
2
Western Blot Analysis of Neuronal Signaling
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RGC-5 lysates of were prepared with a lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.5% NP-40 and protease inhibitor cocktail (Invitrogen, USA). The total protein were then separated on 10% SDS-PAGE gel and transferred to the nitrocellulose membranes. We used primary antibodies against, Thy-1 (1:2000, Santa Cruz Biotechnology, USA), ERK1/2 (1:1000, Cell Signaling Technology, USA), phospho-ERK1/2 (pERK1/2, 1:2000, Cell Signaling Technology, USA), PI3k3 /serine-threonine kinase (AKT, 1:1000, Cell Signaling Technology, USA), phosphor-AKT (pAKT, 1:1000, Cell Signaling Technology, USA), TrkB (1:1000, Cell Signaling Technology, USA), phosphorylated TrkB (1:200, Cell Signaling Technology, USA) and IGF1R (1:200, Santa Cruz Biotechnology, USA). Horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, USA) were used and Actin was used as internal control. The western blots were visualized with an enhanced chemiluminesence system (Amersham Biosciences, USA) according to the manufacturer’s protocol. Band intensities normalized to the total protein immuno-precipitation under each control condition and quantified with an ImageJ software (NIH, USA).
3
Protein Expression Analysis in Breast Cancer Cells
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MDA-MB-231 and MCF-7 cells were lysed by a Tris-based lysis buffer (Thermo Fisher Scientific). Equal amount of protein (20 µg) was drawn from each sample cell lines and separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel (Thermo Fisher Scientific). After transferring proteins into a nitrocellulose membrane, primary antibody against RSU1 (rabbit polyclonal, 1:500; Abcam, USA) was applied to the membrane for 24 h at 4°C. Control membrane was probed by β-actin primary antibody (rabbit polyclonal, 1:2000; Abcam, USA). Then, horseradish peroxidase-conjugated secondary antibody was applied for 2 h at room temperature. Western blot was then visualized using an enhanced chemiluminesence system (Amersham Biosciences, USA).
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