Cells or tissues were lysed in RIPA buffer (9806S) (Cell Signaling Technology) and protein concentration in samples was quantified by BCA kit (Sangon Biotech). Proteins (30 μg) were separated on a 12% SDS‐PAGE and electrotransferred to the treated polyvinyl difluoride membrane (88585) (Thermo Fisher Scientific). After blocking, the membranes were incubated with the primary antibodies against surviving (Cat No: 66495‐1‐Ig), MMP‐2 (Cat No: 66366‐1‐Ig), TfR1 (Cat No: 66180‐1‐Ig), caspase‐3 (Cat No: 66470‐2‐Ig), and β‐actin (Cat No: 66009‐1‐Ig) (Proteintech) overnight at 4°C, then washed thrice with PBS and incubated with the horseradish peroxidase‐conjugated goat anti‐rabbit (Cat No: SA00001‐2) or anti‐mouse IgG (Cat No: SA00001‐1) (Proteintech). Protein bands were detected by a chemiluminescence kit (Order NO: D601039) (Sangon Biotech) and quantitated by ImageJ software.
Chemiluminescence kit
Manufactured by Sangon
Sourced in China
The Chemiluminescence kit is a laboratory equipment designed to detect and measure luminescent signals in biological samples. It provides the necessary reagents and components to enable the chemiluminescent reaction for sensitive and quantitative analysis.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (1 mentions)
Bca kit, by Sangon (1 mentions)
Bicinchoninic acid protein assay kit, by Sangon (1 mentions)
Ripa buffer high, by Solarbio (1 mentions)
Goat anti mouse igg h l, by Proteintech (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
Goat anti rabbit igg h l, by Proteintech (1 mentions)
3 protocols using chemiluminescence kit
1
Protein Expression Analysis in Cell Lysates
2
Western Blot Analysis of Protein Expression
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Washing of the treated cells was carried out in phosphate buffered saline (PBS) and lysed in RIPA buffer (high) (Solarbio, Beijing, China), which contained 1 mM phenylmethylsulfonyl fluoride, Solarbio. Calculation regarding protein concentration was done utilizing the bicinchoninic acid protein assay kit (Sangon Biotech). Equivalent amounts of whole protein extract (30 µg) were electrophoresed on SDS‐polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (pore size: 0.22 µm) using the Yeasen Blot‐transfer unit. After blocking with 5% milk, incubated the membranes overnight with specific primary antibodies against Survivin (Mouse/IgG1; 1:3,000; Proteintech), glyceraldehyde‐3‐phosphate dehydrogenase (Mouse/IgG2b; 1:50,000; Proteintech), His (Mouse/IgG1; 1:10,000; Proteintech), Flag (Rabbit/IgG; 1:10,000; Bioworld), HA (Human influenza hemagglutinin) (Rabbit/IgG; 1:3,000; Bioworld), EGFP (Mouse/IgG1; 1:2000; Leading Biology) at 4°C. The membranes were later incubated with horseradish peroxidase‐conjugated secondary antibody (Goat Anti‐Rabbit IgG (H+L) or Goat Anti‐Mouse IgG (H+L); 1:6000; Proteintech) for 2 h on a horizontal oscillator at room temperature. Products were generated on film by the chemiluminescence kit purchased from Sangon Biotech and quantitated by ImageJ software.32 , 33 , 39
3
Western Blot Protein Extraction
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Total protein from prepared tissues was extracted using RIPA lysis buffer, and the protein concentration was determined using the Bradford assay according to the manufacturer’s instructions. A balanced protein was electrophoresed by SDS-PAGE at 150 V for 100 minutes. Then, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane according to the wet transfer method (400 mA, 60 minutes). The PVDF membrane was blocked using evaporated milk and incubated overnight at 4°C with the primary antibody (1:1,000). The PVDF membrane was then incubated for 1 hour at room temperature with the secondary antibody (1:10,000). Lastly, the protein band was developed on the film using a chemiluminescence kit (Sangon Biotech, Shanghai, China).
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