At 72 h after transfecting with miRNA, GC cells were lysed in RIPA buffer added 1 mm PMSF. Approximately 100 μg of total protein was electrophoresed through 10% SDS/PAGE and was then transferred to a poly(vinylidene difluoride) (PVDF) membrane (Millipore, Boston, MA, USA). After blocking with 5% skimmed milk at 4 °C for 1 h, the membrane was incubated with CCND1 antibody (1 : 1000; Proteintech, Wuhan, China) and GAPDH (1:1000; Proteintech) at 4 °C overnight. The PVDF membrane was then washed and incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody (1 : 10 000; Earthox San Francisco, CA, USA) for 1.5 h at room temperature. Detection was performed by using a SuperLumia ECL HRP Substrate Kit (Abbkine, Wuhan, China). The defined sections of the film were scanned for image capture and quantified using adobe photoshop software (Adobe Systems Incorporated, San Jose, CA, USA) and imagej software (Bio‐Rad).
Superlumia ecl hrp substrate kit
Manufactured by Abbkine
Sourced in China, United States
The SuperLumia ECL HRP Substrate Kit is a solution designed for chemiluminescent detection of horseradish peroxidase (HRP)-labeled biomolecules. The kit includes the necessary components to produce a luminescent signal when HRP reacts with the provided substrate.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated secondary antibody, by EarthOx (3 mentions)
Ccnd1 antibody, by Proteintech (2 mentions)
Gapdh, by Proteintech (2 mentions)
Imagej software, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by EarthOx (1 mentions)
β actin 66009 1 ig, by Proteintech (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
P iκb, by Cell Signaling Technology (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
P src, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Gp91phox, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
8 protocols using superlumia ecl hrp substrate kit
1
Western Blot Analysis of CCND1 in GC Cells
2
Western Blot Analysis of Gastric Cancer Proteins
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Gastric cancer cells were lysed in RIPA buffer with 1 mM PMSF. Total protein (10-100 μg) was electrophoresed through 10% SDS polyacrylamide gels and was then transferred to a PVDF membrane. After blocking with skim milk at 4 °C for 1h, the membrane was incubated with primary antibody at 4°C overnights. The blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10000, Earthox) for 1.5 h at room temperature. Detection was performed using a SuperLumia ECL HRP Substrate Kit (Abbkine) and visualized using a Bio-Rad Imaging System (USA). The primary antibodies used in this study were ILF2 (14714-1-AP, 1:5000, Proteintech, Wuhan, China), ILF3 (13099-1-AP, 1:5000, Proteintech), ELF3 (HPA003316, 1:5000, Sigma, USA), β-Actin (66009-1-Ig, 1:10000, Proteintech), TARBP2 (15753-1-Ig, 1:100, Proteintech), SNAI2 (12129-1-AP, 1:1000, Proteintech), SNAI1 (13099-1-AP, 1:500, Proteintech), YBX1 (20339-1-AP, 1:1000, Proteintech).
3
Investigating Cellular Signaling Pathways
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ALO (purity: 99.8%) was purchased from Selleck Chemicals (Houston, TX, USA). N-acetyl-L-cysteine (NAC) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The Super Lumia ECL HRP substrate kit was purchased from Abbkine Inc (Wuhan, China). The EdU proliferation detection kit was purchased from RiboBio Co., Ltd. (Guangzhou, China). The Cell Counting Kit-8 (CCK-8) was obtained from KeyGen Biotech Co., Ltd. (Nanjing, China). The ROS assay Kit was purchased from the Beyotime Institute of Biotechnology (Haimen,China). Primary antibodies against β-actin, GAPDH, p-Src, p-Stat3, p-Akt, Akt, p-mTOR, mTOR, p-P70S6K, P70S6K, p-S6, S6, p-IκB, and gp91phox were all purchased from Cell Signalling Technology (Beverly, MA, USA). The p47phox and p65 primary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The p22phox primary antibody was purchased from ABclonal Biotechnology Co., Ltd (Hubei, China). Secondary antibodies coupled to the IRDye800 fluorophore for use with the Odyssey Infrared Imaging System were purchased from LI-COR Biosciences (Lincoln, NE, USA).Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were obtained from Cell Signalling Technology (Beverly, MA, USA).
4
Quantitative Analysis of CCND1 Expression
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After 72h transfected with miRNA, Gastric cancer cells were lysed in RIPA buffer added 1 mM PMSF.
Approximately 100 μg of total protein was electropharesed through 10% SDS polyacrylamide gels and were then transferred to a PVDF membrane (Millipone). After blocking with 5% skimmed milk at 4 °C for 1h, the membrane was incubated with CCND1 antibody (1:1000, Proteintech) and GAPDH (1:1000, Proteintech) at 4℃ overnights. The blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10000, Earthox) for 1.5 h at room temperature. Detection was performed by using a SuperLumia ECL HRP Substrate Kit (Abbkine). The de ned sections of the lm were scanned for image capture and quanti cation using Adobe Photoshop software (Adobe Systems Incorporated, USA) and Image J software (Bio-rad, USA).
Approximately 100 μg of total protein was electropharesed through 10% SDS polyacrylamide gels and were then transferred to a PVDF membrane (Millipone). After blocking with 5% skimmed milk at 4 °C for 1h, the membrane was incubated with CCND1 antibody (1:1000, Proteintech) and GAPDH (1:1000, Proteintech) at 4℃ overnights. The blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10000, Earthox) for 1.5 h at room temperature. Detection was performed by using a SuperLumia ECL HRP Substrate Kit (Abbkine). The de ned sections of the lm were scanned for image capture and quanti cation using Adobe Photoshop software (Adobe Systems Incorporated, USA) and Image J software (Bio-rad, USA).
5
Protein Extraction and Analysis from Cell Supernatants
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After stimulation and J114 treatment as indicated, supernatants were collected for the preparation of soluble protein from the cell culture medium. Supernatants were centrifuged at 13,000 revolutions per minute (rpm) for 15 minutes at 4°C to remove cellular debris. Organic solvents (chloroform-methanol) were used to extract protein from the supernatants. The protein in cell lysates was extracted, separated by 12% SDS‒PAGE and transferred to PVDF membranes. The membrane was blocked and incubated with the indicated primary antibody at 4°C overnight, followed by incubation with the appropriate HRP-conjugated secondary antibody for 1 hour at 37°C. The membranes were washed with TBST and subsequently imaged using a Super Lumia ECL HRP substrate kit (Abbkine).
6
Protein Expression Analysis Protocol
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Cells were lysed by RIPA150 lysis buffer as previously described [19 (link)]. Protein samples were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes with protein were blocked with 5%(w/v) skim milk at room temperature for 1 h. Then incubated with primary antibodies at 4℃ for overnight, followed by 1 h incubation with Horseradish Peroxidase (HRP) -conjugated secondary antibodies at room temperature. After 3 times washing with 1 × TBST buffer, SuperLumia ECL HRP Substrate Kit (K22020, Abbkine, Shanghai, China) was used for signal detection.
Primary antibodies are listed as below: GAPDH(60004-1-Ig, Proteintech, Wuhan, China), MGMT(ER40104, HUABIO, Hangzhou, China), γ-H2AX(Ser139)(ET1602-2, HUABIO, Hangzhou, China), caspase-3(9662, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-3(ab32042, Abcam, Cambridge, UK).
Primary antibodies are listed as below: GAPDH(60004-1-Ig, Proteintech, Wuhan, China), MGMT(ER40104, HUABIO, Hangzhou, China), γ-H2AX(Ser139)(ET1602-2, HUABIO, Hangzhou, China), caspase-3(9662, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-3(ab32042, Abcam, Cambridge, UK).
7
Western Blot Analysis of VAMP7 in Gastric Cancer
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Gastric cancer cells were lysed in RIPA buffer added 1 mM PMSF. Approximately 100 μg of total protein was electrophoresed through 10% SDS polyacrylamide gels and were then transferred to a PVDF membrane. After blocking with 5% skimmed milk at 4 °C for 1 h, the membrane was incubated with primary antibody at 4 °C overnights. The blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000, Earthox) for 1.5 h at room temperature. Detection was performed by using a SuperLumia ECL HRP Substrate Kit (Abbkine) and visualized using a Bio-Rad Imaging System (USA). The VAMP7 antibody used in this study was purchased from ABclonal (A18698, Wuhan, China).
8
Optimized Western Blotting Assay
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The western blotting assay was performed as previously described [45 (link)]. The antibodies of AKT (60203-2-Ig), p-AKT (28731-1-AP), p-PI3K (AP0854) and PI3K (20584-1-AP) were purchased from Abclonal company (Wuhan, China) and Proteintech company (Wuhan, China). Briefly, cells were lysed in RIPA buffer added 1 mM PMSF. Approximately 50–100 µg of total protein was electrophoresed through 10% SDS polyacrylamide gels and were then transferred to a PVDF membrane. After blocking with 5% skimmed milk at 4 °C for 1 h, the membrane was incubated with primary antibody at 4 °C overnights. The blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10,000, Earthox) for 1.5 h at room temperature. Detection was performed by using a SuperLumia ECL HRP Substrate Kit (Abbkine) and visualized using a Bio-Rad Imaging System (USA).
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