Fresh HC and PFC samples were collected (n = 6 in each group) to measure the protein levels of apoptotic factors. The tissues were homogenized in a lysis buffer and centrifuged at 15,000 rpm (10 min, at 4 °C). The total protein levels from each experiment were evaluated using a total protein kit (Micro, Sigma, USA). After protein denaturation, we exposed 5 μg of protein from each experiment to 10% SDS-PAGE and transferred it to a polyvinylidene difluoride (PVDF) transfer membrane (Sigma, USA). Then, the blots were blocked with blocking buffer (5% skimmed milk powder in PBS) at room temperature (20–22 °C) for 2 h. Additionally, they were incubated with antibodies against Bax (1/1000 v/v, Cat: ab216494, Abcam, Germany) and Bcl2 (1:1000, Cat: ab196495, Abcam, Germany) overnight at 4 °C and washed four times with 0.1% Tween 20 in PBS. The samples were subsequently incubated with HRP-conjugated secondary antibody (1/10,000 v/v) at room temperature for 1 h. Protein bands were visualized with a luminescent substrate solution (Sigma, USA) and quantified using ImageJ (NIH, USA). GAPDH (Cat: ab181602, Abcam, Germany) was used for normalization. Images of blots with adequate length and membrane edges could not be provided because the blots were cut prior to hybridisation with antibodies and scanned at inside of blots, respectively.
Luminescent substrate solution
Manufactured by Merck Group
Sourced in United States
Luminescent substrate solution is a laboratory reagent used to measure the activity of enzymes that produce light, such as luciferase. The solution contains the necessary compounds to facilitate a luminescent reaction, allowing for the quantification of the target enzyme. This product is designed for use in various applications, including cell-based assays, reporter gene studies, and in vitro enzyme activity measurements.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene di uoride transfer membranes, by Merck Group (2 mentions)
Speci c primary antibodies, by Novus Biologicals (2 mentions)
Horseradish peroxidase hrp conjugated anti rabbit secondary antibody, by Abcam (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Abcam (1 mentions)
Total protein kit, by Merck Group (1 mentions)
3 protocols using luminescent substrate solution
1
Apoptotic Factors Protein Quantification
2
Western Blot Analysis of Inflammasome Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The isolated hippocampal samples were used for western blot (n = 4 in each group) to determine the synthesis of in ammasome proteins. The samples were lysed via a lysis buffer (RIPA) and centrifuged. Total Protein Kit, Micro (Sigma, USA) was used to detect the total protein concentration. After protein denaturation, 5 μg of protein was loaded on 10% SDS-PAGE, and separated proteins were put on polyvinylidene di uoride transfer membranes (Sigma, USA) and then incubated for one hour with speci c primary antibodies (Novus Biologicals, USA). Then, the procedure was followed by incubation of Membranes for one hour with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Germany). Protein bands were detected by the luminescent substrate solution (Sigma, USA). Finally, to quantify the speci c bands, ImageJ software (NIH, USA) was used. GAPDH (Thermoscienti c, USA) was used for normalization [33] .
3
Hippocampal Inflammasome Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Using western blot technique, the isolated hippocampal samples were used (n = 4 in each group) to determine the synthesis of in ammasome proteins. The samples were lysed via a lysis buffer (RIPA) and centrifuged. Total Protein Kit, Micro (Sigma, USA) was used to detect the total protein concentration. After protein denaturation, 5 μg of protein were loaded on 10% SDS-PAGE, and separated proteins were put on polyvinylidene di uoride transfer membranes (Sigma, USA) and then incubated for one hour with speci c primary antibodies (Novus Biologicals, USA). Then, the procedure was followed by incubation of membranes for one hour with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Germany). Protein bands were detected by the luminescent substrate solution (Sigma, USA). Finally, to quantify the speci c bands, ImageJ software (NIH, USA) was used. GAPDH (Thermoscienti c, USA) was used for normalization [33] .
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!