Briefly, cells were gently lysed by incubation in ice-cold RIPA buffer (Beyotime, China) containing protease inhibitors (Beyotime, China) for 30 min followed by centrifugation at 12,000 × g for 15 min; then, the supernatant was collected. The blots were probed with rabbit anti-human β-actin (Multi Science; 1:5000), rabbit anti-human Fibulin2 (Abcam, ab234993; 1:2000), rabbit anti-human Notch2 (Abcam, ab245325; 1:2000), rabbit anti-human Hes1 (Abcam, ab108937; 1:1000), rabbit anti-human Col1a1 (Abcam, ab260043; 1:1000), and rabbit anti-human Runx2 antibodies (Abcam, ab236639; 1:1000). The secondary antibody was peroxidase-conjugated goat anti-rabbit Ig (Abcam, ab205718; 1:5000). Protein ladder(10-180KD) was provided from Biosynthesis Biotechnology Inc. (Beijing, China). Densitometry was performed with ImageJ software.
Ab245325
Manufactured by Abcam
Sourced in United States
Ab245325 is a laboratory equipment product offered by Abcam. It is a versatile device designed for general laboratory applications. The core function of this product is to facilitate standard laboratory procedures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Solarbio (2 mentions)
Ab236639, by Abcam (1 mentions)
Ab260043, by Abcam (1 mentions)
Ab108937, by Abcam (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab234993, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab92544, by Abcam (1 mentions)
Ab68183, by Abcam (1 mentions)
Enhanced chemiluminescent substrate kit, by Merck Group (1 mentions)
Ab260038, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab8227, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Ab240753, by Abcam (1 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Ab184742, by Abcam (1 mentions)
4 protocols using ab245325
1
Protein Expression Analysis in Cells
2
Protein Extraction and Immunoblotting in BT474/OX and MCF-7/OX Cells
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Extraction of total protein from BT474/OX or MCF-7/OX cells was conducted with RIPA buffer (Sigma-Aldrich). Then, total protein quantitation was performed by Bradford method. Then, protein samples were subjected to SDS-PAGE and transferred onto PVDF membranes (Roche, Switzerland). After blocked with 5% skim milk, the PVDF membranes were cultivated with primary antibodies against MRP1 (1:1000; ab260038; Abcam), MDR1 (1:1000; #13,978; Cell signaling technology), LRP1 (1:1000; ab92544; Abcam), Notch2 (1:1000; ab245325; Abcam), GSK-3β (1:1000; #12,456; Cell signaling technology), β-catenin (1:1000; ab68183; Abcam), or β-actin (1:1000; ab8227; Abcam) at 4°C overnight and incubated with secondary antibody for 2 hours at room temperature. The protein bands were visualized with an enhanced chemiluminescent substrate kit (Millipore). The proteins were quantified using Quantity One software (Bio-Rad Laboratories, USA).
3
NEDD4L-Notch2 Protein Interaction
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A549 cells were treated with RIPA buffer (Solarbio, Shanghai, China) containing phenylmethylsulfonyl fluoride and incubated with NEDD4L antibody (1:200, ab240753; Abcam, Cambridge, MA, USA) for 14 h at 4 °C. Protein A/G magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) were added into IP mixture for 12 h at 4 °C. After washing with PBS, the precipitates were assessed through western blot analysis using Notch2 antibody (1:3,000, ab245325; Abcam, Cambridge, MA, USA).
4
Quantifying Notch Receptor Expression in LUAD Cells
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Total proteins were obtained from LUAD cells with RIPA buffer (Solarbio, Shanghai, China) and quantified by BCA protein quantification kit (Solarbio, Shanghai, China). Approximately 40 µg of total protein were separated through a 10% SDS-PAGE and were then transferred to PVDF membranes. Membranes were next incubated with the specific primary antibody against Notch1 (1:1,000, ab280898; Abcam, Cambridge, MA, USA), Notch2 (1:3,000, ab245325; Abcam, Cambridge, MA, USA), Notch3 (1:600, ab252845; Abcam, Cambridge, MA, USA), Notch4 (1:600, ab184742; Abcam, Cambridge, MA, USA), and ubiquitin (1:1,500, ab134953; Abcam, Cambridge, MA, USA) for 1 h at room temperature (RT). After washing with TBST five times, membranes were incubated with HRP-coupled anti-rabbit or mouse secondary antibody (1:6,000) for 1 h at RT. Immunoblots were visualized with the chemiluminescence detection system.
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