I followed the exact protocol for kidney, liver samples and it worked. But it is not working for brain samples. I extracted the histone proteins using pre-lysis, lysis, and balanced buffer from Bio-Rad. Then checked the protein concentration followed by SDS-PAGE (80V for 1.5 hours). Then I did the transfer (0.18A for 1.5 hours) on PVDF. I used H3K9BHB primary antibody (1:1000)dilution and kept in in cooler for 12-16 hours followed by secondary antibody (Anti-goat Rabbit 1:10000 dilution) for 2 hours. I developed the blots using LICOR's fluorescence-based detection.
Hi Sachin,
This can be as results of lack reduction, insufficient heating, unstable voltage during gel electrophoresis or high temperature resulted from a high current.
Can you provide with some information about your experiment protocol? It is better also to add a picture of your gel after staining with coammasie if it is applicable.
Best..
Maybe there is a lipid contamination which may interfere with SDS-PaGE. I guess that lipids are more abundant in brain extract than in kidney or liver.
Thank you Mohamed Khashan . I was also suspecting the same but I didn't see any papers that talk about delipidation measures while working on brain samples for the SDS-PAGE. Do you have any idea how I can do it Dr. Tomasz Fraczyk ?
Dear Sachin Aryal
I do not have experience in delipidation of protein samples, but extraction of lipids with organic solvents comes to my mind. An example of the protocol:
https://cyberlipid.gerli.com/techniques-of-analysis/extraction-handling-of-extracts/delipidation-of-plasma/
The other way is to delipidate using solid material binding lipids, like in:
Article A solvent-free delipidation method for functional validation...
Unfortunately, I cannot say which approach is better for your samples.
Kind regards
Take a look at;
https://www.bio-rad.com/en-tr/product/readyprep-2-d-cleanup-kit?ID=MGSKJME8Z
Thank you, Dr. İsmail Emir Akyildiz . This kit should work for my case.
To remove lipids from protein samples before performing SDS-PAGE, you can follow these steps:
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
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