On the basis of the manufacturer’s guidelines, a standard BCA test was executed to determine the protein concentration in cell lysate. After isolation via SDS-PAGE (10%) electrophoresis, the proteins were transferred to PVDF membranes at 4 °C and sealed with skim milk (5%) in TBST for 1 h. These membranes were incubated with anti-GAPDH or anti-UPF1 antibody (Cell Signaling, USA) overnight at 4 °C. The membranes were washed three times with TBST and incubated with secondary antibody at indoor temperature for 1 h. A Phototope-horseradish peroxidase Western blot detection kit (Cell Signaling Technology, Danvers, MA, USA) was applied to detect the expression of proteins. The UPF1 protein expression levels were normalized to that of GAPDH by calculating the relative expression levels.
Phototope horseradish peroxidase western blot detection kit
Manufactured by Cell Signaling Technology
Sourced in United States
The Phototope-horseradish peroxidase Western blot detection kit is a laboratory equipment product designed for the detection of target proteins in Western blot analysis. The kit contains reagents necessary for the visualization of protein bands on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Phospho akt thr308, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 9 antibody, by Cell Signaling Technology (1 mentions)
Hybond enhanced chemiluminescence, by Cytiva (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Miniprotein 3 system, by Bio-Rad (1 mentions)
Monoclonal antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Ab93434, by Abcam (1 mentions)
6 protocols using phototope horseradish peroxidase western blot detection kit
1
Quantification of UPF1 Protein Levels
2
Western Blot Analysis of Cellular Proteins
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Total protein (60 μg) extracted from cells or tissue samples was resolved by SDS-PAGE and transferred to PVDF membrane (Millipore). Antibody against BACH1 (A303-057A) was from Bethyl Laboratories while antibodies against other proteins were from Abcam (HO-1, ab52947; ERK1/2, ab36991; phosphorylated ERK1/2, ab76299; NRF2, ab62352; OCT4, ab181557; ABCG2, ab207732; ALDH1, ab52492 and TRA-1-60, ab16288), Santa-Cruz (HIF1A, sc-10790; VEGF, sc-152 and β-ACTIN, sc-47778) and Cell Signaling Technology (AKT, #9272; phospho-AKTThr308, #9275; phospho-AKTSer473, #9271; PTEN, #9559; eNOS, #9586; phospho-eNOSSer1177, #9570; E-CADHERIN, #3195; VIMENTIN, #5741; ZO-1, #8193; ZEB1, #3396 and SLUG, #9585). The membranes were incubated with the primary antibody and visualized with a Phototope-horseradish peroxidase Western Blot Detection kit (Cell Signaling Technology).
3
Protein Expression Analysis of BGC-823 and SGC-7901 Cell Lines
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Protein was isolated from BGC-823 and SGC-7901 cell lines as previously stated [19 (link)].
Forty micrograms of total protein s were run on a 14.7% polyacrylamide gel, and then transferred to polyvinylidene difluoride membranes (Hybondenhanced chemiluminescence; Amersham Pharmacia Biotech). The antibodies against FBWX7(1:1000) and β-actin (1:1000) were purchased from Abcam (Abcam, Cambridge, MA, USA). The antibodies against Caspase-9 antibody (1:1000) and Caspase-3 (1:1000) were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). The protein was measured with a Phototope–horseradish peroxidase Western blot detection kit (Cell Signaling Technology, Inc.), and β-actin was treated as an internal control.
Forty micrograms of total protein s were run on a 14.7% polyacrylamide gel, and then transferred to polyvinylidene difluoride membranes (Hybondenhanced chemiluminescence; Amersham Pharmacia Biotech). The antibodies against FBWX7(1:1000) and β-actin (1:1000) were purchased from Abcam (Abcam, Cambridge, MA, USA). The antibodies against Caspase-9 antibody (1:1000) and Caspase-3 (1:1000) were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). The protein was measured with a Phototope–horseradish peroxidase Western blot detection kit (Cell Signaling Technology, Inc.), and β-actin was treated as an internal control.
4
Western Blot Analysis of MEK5 Protein
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Protein samples (20 μg) were separated by 10 % acrylamide gel using a Bio-Rad Mini-Protein III system (100 V for 2 h) and then transferred to PVDF membranes in 200 mA for 50 min in transfer buffer. The membranes were blocked for 90 min with 5 % skimmed milk powder in 0.05 % TBS-T at room temperature. The monoclonal antibody against MEK5 was purchased from BD Transduction Laboratories (San Diego, CA, USA), and the monoclonal antibody against β-actin was purchased from Santa Cruz Biotechnology. The membranes were then incubated overnight at 4 °C with primary antibodies in 2 % BSA dissolved in TBS-T (1:500 dilution), and the proteins were detected with a Phototope-horseradish peroxidase Western blot detection kit (Cell Signaling Technology, Inc). Protein expression levels were normalized to that of β-actin by calculating the relative expression levels.
5
Protein Expression Analysis in Tissues
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Total proteins extracted from tissue samples or cell lines were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). Antibody against TIGAR (ab62533), GLS (ab93434) or GLUD1 (ab34786) from Abcam, antibody against phosphorylated AMPKα at Thr172 (2535), AMPKα (5831) or ASCT2 (SLC1A5; 3545) from CST and antibody against β-ACTIN (sc-47778) from Santa-Cruz were used. The membranes were incubated with the primary antibody and visualized with a Phototope-Horseradish Peroxidase Western Blot detection kit (Cell Signaling Technology). The protein bands were quantified by gray scanning.
6
Protein Expression Analysis of PM2.5 Exposure
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Western blotting assay was used to detect the protein expression levels of interest genes in HBE cells exposed to PM2.5 extract or cells transfected with expression plasmids containing the interest genes. Protein extract prepared by detergent lysis was subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore, Temecula, CA). Antibodies against SLC30A1 (ab110383, Abcam, Cambridge, MA), SERPINB2 (16035-1-AP, ProteinTech, Chicago, IL), AKR1C1 (7660-1, Epitomics, Burlingame, CA) or β-ACTIN (20536-1-AP, ProteinTech) were used. The membranes were incubated overnight at 4°C with the primary antibody and the proteins were then detected with a Phototope-horseradish peroxidase Western blot detection kit (Cell Signaling Technology).
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