For western blots, 2 × 105 OVCAR-3 or A2780 cells per well were plated in a 6-well plate for 24 h. Subsequently, the cells were incubated with the appropriate drug for distinct time periods and the western blots were performed as previously described [36 (link)] using the following antibodies: Capsese-3 (Cell Signaling Technology, USA, #9661), PARP (Cell Signaling Technology, USA, #9542), Microtubule-associated protein 1 light chain 3 (LC3) (Sigma, USA, #L7543), p62/SQSTM1/SQSTM1 (Cell Signaling Technology, USA, #5114), ATG5 (Cell Signaling Technology, USA, #9980), ATG7 (Cell Signaling Technology, USA, #8558), p-AKT (Cell Signaling Technology, USA, #9271), AKT (Cell Signaling Technology, USA, #9272), BAX (Proteintech Group, USA, #23931-1-AP), Bcl-2 (Proteintech Group, USA, #12789-1-AP), and Mcl-1 (Proteintech Group, USA, #16225-1-AP), p-p70S6K (Thr389) (Sigma, USA, #MABS82), p70S6K (Sigma, USA, #06-926), p-mTOR (S2448) (Abcam, USA, #ab109268), -S6 Ribosomal (Cell Signaling Technology, USA, #5364), S6 Ribosomal (Cell Signaling Technology, USA, #2317), mTOR (Abcam, USA, #ab2732), and β-actin (Origene, USA, #TA811000), peroxidase-linked anti-rabbit antibody (Cell Signaling Technology, Danvers, USA, #7074) or peroxidase-linked anti-mouse antibody (Sigma-Aldrich, St. Louis, USA, #A9044).
Peroxidase linked anti mouse antibody
Manufactured by Merck Group
Sourced in United States
The Peroxidase-linked anti-mouse antibody is a laboratory reagent used for the detection and quantification of mouse-derived proteins or antigens in various experimental procedures. The antibody is conjugated with the enzyme peroxidase, which can catalyze a color-producing reaction, allowing for the visualization and measurement of the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase linked anti rabbit antibody, by Cell Signaling Technology (2 mentions)
Mouse anti β actin antibody, by Merck Group (2 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Mcl 1, by Proteintech (1 mentions)
Microtubule associated protein 1 light chain 3, by Merck Group (1 mentions)
P70s6k, by Merck Group (1 mentions)
S6 ribosomal, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
β actin, by OriGene (1 mentions)
P mtor, by Abcam (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Dmem high glucose medium, by Harvard Bioscience (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
3 protocols using peroxidase linked anti mouse antibody
1
Western Blot Analysis of Apoptosis and Autophagy
2
Western Blot Analysis of Apoptosis Markers
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The cells were seeded on 6 well plates in DMEM high glucose medium (Biochrom GmbH) supplemented with 10% fetal calf serum. On the following day the cells covered about 30% of the well surface and were incubated for another 24 or 48 hours with control medium or medium containing therapeutic agents (as described above). After separating the cell lysates on SDS polyacryl gels the proteins were transferred to a polyvinyldifluoride membrane (Immobilon-P; Millipore, Eschborn, Germany) as described previously 10 . The membranes were blocked with 2.5 % (wt/vol.) BSA and incubated overnight at 4 °C with rabbit-anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA, code 9661, dilution: 1000x) or rb-anti PARP (Cell Signaling, Danvers, MA, USA, code 9542, dilution: 1000x) followed by incubation with a secondary peroxidase linked anti-rabbit (Cell Signaling, code 7074, dilution: 2000x for cleaved caspase 3 and 20000x for PARP Western Blot). For analysis of β-actin production, membranes were stripped, blocked by 2.5 % (wt/vol.) BSA and incubated with mouse anti-β-actin antibody (Sigma-Aldrich, code A5441, dilution: 20000x) followed by peroxidase-linked anti-mouse antibody (Sigma-Aldrich, USA; code A9044, dilution: 60000x).
3
Quantifying Autophagy and Apoptosis Markers
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For western blots, 2.4 × 105 cells per well were plated in a 6-well plate. After 24 h, the cells were treated with the appropriate drug for distinct time periods. In addition, in experiments analyzing autophagic flux with CQ, the cells were treated for a maximum of 6 h just before the harvest of the cells. The western blots were performed as previously described [16] (link) using the following antibodies: rabbit anti-microtubule-associated protein 1 light chain 3 (LC3, Sigma-Aldrich, St. Louis, USA, code L7543, dilution: 1000×), rabbit anti-p62/SQSTM1 (p62, Abcam, Cambridge, UK, code ab 109012, dilution: 8000×), rabbit anti-cleaved caspase 3 (Cell Signaling, Danvers, USA, code 9661, dilution: 1000×), mouse anti-β-actin antibody (Sigma-Aldrich, St. Louis, USA, code A5441, dilution: 20000×), peroxidase-linked anti-rabbit antibody (Cell Signaling, Danvers, USA, code 7074, dilution: 10000×) or peroxidase-linked anti-mouse antibody (Sigma-Aldrich, St. Louis, USA, code A9044, dilution: 60000×). The ratios of LC3II/β-actin and p62/β-actin were determined using a Chemi-Doc XRS System (Bio-Rad Laboratories, Munich, Germany) and are presented as the mean ± standard deviation (SD).
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