Protein concentrations were determined using the BCA protein assay kit (Beyotime, Beijing, China). The protein samples were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked with 5% non-fat milk for 1 h and incubated with the following primary antibodies overnight at 4 °C: anti-Bax (1:1000, Abcam), anti-Bcl-2 (1:1000, Abcam), anti-cleaved caspase-3 (1:1000, Abcam), anti-GSDME-N (1:1000, Abcam), anti-cleaved caspase-1 (1:1000, Abcam), anti-p-EGFR (1:1000, Abcam), anti-EGFR (1:1000, Abcam), and anti-β-actin (1:1000, Abcam). The following day, the membranes were incubated with the horseradish peroxidase-conjugated IgG secondary antibody (1:5000, Abcam) at room temperature for 1 h. Finally, the blots were visualized using the ECL chemiluminescent substrate reagent (Thermo Fisher Scientific).
Ecl chemiluminescent substrate reagent
Manufactured by Thermo Fisher Scientific
ECL chemiluminescent substrate reagent is a laboratory product designed for the detection and quantification of proteins in Western blot analysis. It generates a luminescent signal when it reacts with the horseradish peroxidase (HRP) enzyme, which is commonly used to label antibodies in Western blot experiments. The reagent can be used to visualize the presence and relative abundance of target proteins on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Anti egfr, by Abcam (1 mentions)
Anti cleaved caspase 1, by Abcam (1 mentions)
Anti p egfr, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti bax, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
2 protocols using ecl chemiluminescent substrate reagent
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot for TPH2 Overexpression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Over expression of TPH2 was validated by standard western blot procedure (Fig. S1† ) followed by chemiluminescent imaging.62 (link) Briefly, protein was extracted from cells using a Urea-CHAPS buffer (8 M Urea, 1% CHAPS, and 20 mM HEPES, pH 8.0) and run on a Bis–Tris resolving gel. Proteins were transferred to a polyvinylidene difluoride membrane (PVDF), blocked with 5% milk and probed with antibodies to TPH2 (Abcam ab111828) and GAPDH (Proteintech 60004) overnight at 4 °C. HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were then incubated for 2 hours at room temperature with ECL Chemiluminescent Substrate Reagent (Thermo Scientific) applied for detection.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!