Cell lysates were equally loaded onto 4% to 15% SDS-PAGE (Bio-Rad), electrophoresed, and transferred to enhanced chemiluminescence–nitrocellulose membranes (Bio-Rad). The membranes were stained with 0.2% Ponceau S red to ensure equal protein loading. After blocking with 5% nonfat milk in TBS, the membranes were incubated with antibodies against FPN1 (Abcam), STAT3, p-STAT 3, MCL-1, ERK1/2, p-RRK, JNK, p-JNK, p38, p-p38, NfkB-pNFkB, AKT, p-AKT, BCL2, BCL-XL, BAX, BAK, BIM, NOXA, BID, and PUMA (Cell Signaling Technology) overnight at 4°C, followed by horseradish peroxidase–linked secondary antibody (Cell Signaling Technology) for 1 hour at room temperature. Detection was developed by Immobilon Western ECL substrate (Millipore), according to the manufacturer’s instructions. β-actin (Cell Signaling Technology) was used as an internal control.
Immobilon western ecl substrate
Manufactured by Merck Group
The Immobilon Western ECL substrate is a chemiluminescent detection reagent used for Western blot analysis. It is designed to detect horseradish peroxidase (HRP)-labeled target proteins on a membrane. The substrate generates a luminescent signal proportional to the amount of target protein present, which can be captured and quantified using an appropriate imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Stat3, by Abcam (1 mentions)
P stat3, by Abcam (1 mentions)
F7425, by Merck Group (1 mentions)
Skimmed milk powder, by Merck Group (1 mentions)
Casein solution, by Merck Group (1 mentions)
Anti mouse igg hrp, by Agilent Technologies (1 mentions)
Odyssey fc device, by LI COR (1 mentions)
Anti rabbit irdye 680, by LI COR (1 mentions)
Odyssey clx scanner, by LI COR (1 mentions)
3 protocols using immobilon western ecl substrate
1
Western Blot Analysis of Cellular Signaling Proteins
2
Western Blot Analysis of Yeast Proteins
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Protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes by wet electroblotting following the manufacturer’s protocol. Western blotting signals were developed using immobilon Western ECL Substrate (Millipore) and visualized by chemiluminescence system (Tanon, T5200Multi). Same amounts of whole cell extracts from the yeast strains were loaded for analyzing the expression levels of Flag-tagged Rpf2p and HA-tagged Noc2 proteins and equal loading was controlled by determination of the protein level of GAPDH. The pre-ribosome purification samples containing equal amounts of Flag-tagged Rpf2p were loaded for investigating the assembled ability of Noc2 proteins into pre-ribosomes. Western blot analysis was performed using the following antibodies: anti-Flag rabbit polyclonal antibody (Sigma Cat No. F7425, 1:5000, RRID: AB_439687), anti-HA mouse monoclonal antibody (Sigma Cat No. H3663, 1:10 000, RRID: AB_262051), anti-GAPDH rabbit polyclonal antibody (GeneTex Cat No. GTX100118, 1:5000, RRID: AB_1080976).
3
Western Blot for Protein Detection
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Proteins were separated by 8% or 10% SDS–PAGE and transferred onto PVDF membranes (0.45 mM, #T830.1; Carl Roth). The membranes were blocked in 5% skimmed milk powder (Sigma-Aldrich) or in casein solution (Sigma-Aldrich) for 1 h at RT. Primary antibodies were incubated overnight at 4°C in 5% skimmed milk and 0.1% Tween. Membranes were washed 3× in TBS/0.05% Tween and incubated with secondary antibody for 1 h at RT. Membranes were washed 3× in TBS/0.05% Tween. Proteins were detected either after short incubation in Immobilon Western ECL substrate (#WBKLS0500; Millipore) on an Odyssey FC device (Li-cor Biosciences) or on an Odyssey CLx scanner (Li-cor Biosciences). The following antibodies were used: ADAM1974–123 (#NBP1-69367; Novus), ADAM19218–267 (#ab104800; Abcam), FLAG (#TA50011-100; Origin), PTHR14–54 (#ABIN2776779; Antibodies-Online.de), PTHR152–86 (#ABIN5708269; Antibodies-Online.de), FLAG (#TA50011-100; Origin), HA (#137838; Abcam), polyclonal PTHR1573–593 (Lupp et al, 2010 (link)), anti-mouse IgG-HRP (#P0260; Dako), anti-rabbit IrDye680 (#926-32223; Li-cor Biosciences).
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