Cells and extracellular vesicles were collected in a Laemmli buffer containing Tris-HCl pH 8 (120 mM), glycerol (20% v/v), and sodium dodecyl sulfate (4% m/v). Total proteins were quantified with NanoDrop (absorbance at 280 nm). For kinetics of ∆Raf-1-ER activation, 20 µg of protein extracts was loaded on bilayered SDS-PAGE with 15% acrylamide in the lower half and 8.5% acrylamide in the upper half of the gel under a 4% stacking layer. BLUeye Prestained Protein Ladder (FroggaBio, Concord, ON, USA) was used as a molecular ladder. Proteins were transferred to the nitrocellulose membrane (Biorad, Saint-Laurent, QC, Canada). Primary antibodies used for immunoblotting were anti-estrogen receptor alpha (1:1000, clone F-10, sc-8002, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p44-42 MAPK (Erk1/2) (1:1000, #9102, Cell Signaling Technology, Danvers, MA, USA), anti-Phospho-p44/42 MAPK Erk1/2 (Thr202/Tyr204) (1:1000, clone D13.14.4E, #4370, Cell Signaling Technology), anti-Histone H3 (1:2000, ab1791, Abcam, Cambridge, UK), anti-Phospho-histone H3 (Ser10) (1:1000, #06-570, Millipore), anti-α-Tubulin (1:10,000, clone B-5-1-2, T6074, Sigma). Secondary antibodies coupled to peroxidase (Biorad) and ECL detection reagent were used to reveal the signals.
Blueye prestained protein ladder
Manufactured by FroggaBio
Sourced in United States
The BLUeye Prestained Protein Ladder is a molecular weight marker used for the estimation of the molecular weights of proteins in a polyacrylamide gel electrophoresis (PAGE) experiment. It contains a set of pre-stained protein bands with known molecular weights, which can be used as reference points for determining the molecular weights of the target proteins in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Anti phospho histone h3 ser10, by Merck Group (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Anti phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Goat anti rabbit igg secondary antibody, by LI COR (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
A7250, by Merck Group (1 mentions)
2 protocols using blueye prestained protein ladder
1
Immunoblotting of Cellular Proteins
2
PTEN Redox State Analysis
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Whole-cell lysates were prepared from 5x10 6 SeptMBr cells treated with Nacetylcysteine (MilliporeSigma A7250) for 4 hours followed by stimulation/oxidation with 1 mM hydrogen peroxide (Fisher Scientific Canada, Mississauga ON) for 5 minutes. Cells were washed and resuspended in PBS with 10% trichloroacetic acid (MillipreSigma T9159), sonicated, and washed again in 100% acetone. To preserve PTEN protein in its reduced state, protein precipitates were resolubilized in non-reducing lysis buffer (2% SDS, 50 mM Tris-HCl pH 6.8, 10% glycerol, 0.1% bromophenol blue) containing 40 mM N-ethylmaleimide (MilliporeSigma E3876). Samples were denatured at 100°C and separated on 10% SDS-PAGE gels with BLUeye Prestained Protein Ladder (FroggaBio, Concord ON). Protein was then semidry transferred to a PVDF membrane and incubated in Li-Cor Intercept ® Blocking Buffer (Cedarlane, Burlington ON). Immunoblotting was performed with monoclonal anti-PTEN rabbit IgG primary antibody (Cell Signalling Technology 9188S, Danvers MA) at 4°C overnight and goat anti-rabbit IgG secondary antibody (Li-Cor 926-32211) for 1 hour at room temperature.
Blots were visualized with the Odyssey CLx Infrared imaging system at 800 nm (Li-Cor).
Blots were visualized with the Odyssey CLx Infrared imaging system at 800 nm (Li-Cor).
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