The protein samples were separated using precast gels and transferred to the membrane using an iBlot1 gel transfer device (Thermo Fisher Scientific, Waltham, MA, USA). Membranes were blocked in 5% blotting grade blocker made with 1x Tris buffered saline-Tween 20 (TBS-T) buffer for 1 hour. The primary antibody was added and the membrane was left to rock overnight at 4°C. The membrane was incubated with a secondary antibody. For membranes that were stripped, restore western blot stripping buffer was used; none of the membranes in this study were stripped more than three times. The following non-human antibodies were used: P-p38MAPK (1:300, Cell Signaling, T180/Y182), p38MAPK (1:1000, Cell Signaling), actin (Sigma-Aldrich, A5441).
Iblot1 gel transfer device
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBlot1 gel transfer device is a laboratory equipment used for the rapid and efficient transfer of proteins from polyacrylamide gels to membranes for further analysis, such as Western blotting. It utilizes an electrical current to facilitate the transfer process.
Automatically generated - may contain errors
Lab products found in correlation
P38 mapk, by Cell Signaling Technology (2 mentions)
P p38 mapk, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody, by Cell Signaling Technology (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
β actin 13e5, by Cell Signaling Technology (1 mentions)
Vimentin, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
5 protocols using iblot1 gel transfer device
1
Western Blot Protein Analysis
2
Western Blotting Analysis of Protein Signaling
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Western blotting analysis was performed as described previously (24 (link)). Cells were collected 48 hours after administration of LY294002 and bevacizumab and transfection with siRNA VEFR2. Irradiation and administration of olaparib were performed 2 and 8 hours before cell collection, respectively. Total protein was resolved on gradient NuPage 4%–12% Bis-Tris gels (Thermo Fisher Scientific) and transferred to membranes using an iBlot1 Gel Transfer Device (Thermo Fisher Scientific). The membranes were incubated sequentially with primary antibodies at 4°C and horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody (1:10000, Cell Signaling Technology, Beverly, MA) at room temperature with gentle agitation. Positive immunoreactions were detected using the ImmunoStar LD chemiluminescence system (Wako, Tokyo, Japan). Rabbit polyclonal antibody against CRY1 (EPR165; #ab229631; 1:1000) was purchased from Abcam (Cambridge, UK), and rabbit monoclonal antibodies against AKT (11E7; #4685; 1:1000), phosphorylated AKT (Thr308) (244F9; #4056; 1:1000), phosphorylated AKT (Ser473) (D9E; #4060; 1:2000) and β-actin (13E5; #4970; 1:4000) were purchased from Cell Signaling Technology.
3
Protein Extraction and Western Blot Analysis
4
Western Blotting of Ovarian Cancer Proteins
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Western blotting was performed as previously described [7 7 . Yokomizo, R. • Yanaihara, N. • Yamaguchi, N. ... MicroRNA-34a/IL-6R pathway as a potential therapeutic target for ovarian high-grade serous carcinoma Oncotarget. 2019; 10:4880-4893 Crossref PubMed Google Scholar ]. Total protein was resolved on gradient NuPage 4%-12% Bis-Tris gels (Thermo Fisher Scientific) and then transferred onto membranes using an iBlot1 Gel Transfer Device (Thermo Fisher Scientific). The membranes were incubated sequentially with primary antibodies at 4 °C and horseradish peroxidase-conjugated secondary antirabbit or anti-mouse antibody (1:10000, Cell Signaling Technology, Beverly, MA) at room temperature with gentle agitation. Positive immunoreactions were detected using the ImmunoStar LD chemiluminescence system (Wako, Tokyo, Japan). Antibodies against CDK1 (clone EPR165; ab133327; 1:10000) and BRCA1 (clone 6B4; GTX70115; 1:500) were obtained from Abcam (Cambridge, UK) and GeneTex (Irvine, CA), respectively. Rabbit polyclonal antibodies against phosphorylated BRCA1 (Ser1497) (AF8204; 1:1000) and phosphorylated BRCA1 (Ser1189) (A300-003A; 1:2000) were obtained from Affinity Biosciences (Cincinnati, OH) and Bethyl Laboratories (Waltham, MA), respectively. Rabbit monoclonal antibodies for β-actin (13E5; #4970; 1:4000) and HA-tag (C29F4; #3724; 1:1000) were obtained from Cell Signaling Technology.
5
Protein Extraction and Western Blot Analysis
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Decidua and mice tissue were lysed with RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, and 1.0 mM EDTA pH 8.0, 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktail and phenylmethylsulphonyl fluoride. After centrifugation at 10,000 rpm for 20 min, the supernatant was collected. Protein quantification was performed using the Pierce BCA protein assay kit (Thermo Fisher Scientific). The protein samples (N = 3) were separated using precast gels and transferred to the membrane using an iBlot1 gel transfer device (Thermo Fisher Scientific, Waltham, MA, United States). Membranes were blocked in 5% blotting grade blocker made with 1x Tris buffered saline-Tween 20 (TBS-T) buffer for 1 h. The primary antibody was added and the membrane was left to rock overnight at 4°C. The membrane was incubated with a secondary antibody. For membranes that were stripped, restore western blot stripping buffer was used; none of the membranes in this study were stripped more than three times. The following non-human antibodies were used: vimentin (ab92547, Abcam Inc. Cambridge, MA, United States), actin (Sigma-Aldrich, A5441), and STAT3 (SC8019, Santa Cruz, Waltham, MA, United States).
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