Hi im trying to detect by western blot a nuclear protein. I have been trying yo do this with no success until i realised that nuclear proteins may require different way to prepare and detect. Below is how i prepare my cell lysate for western blot detection typically please advise on the centrifugation part to detect nuclear protein.
Remove media from culture dish and add PBS. Remove pbs and add 50ul of lysis buffer into cells. Scrape to detach and then homogenize with syringe. Transfer contents into ependorf tube and centrifuge for 14000 rpm for 15 mins. The supernatant is normally used for western blot detection. Now is there any advise on how to modify this centrifugation step to enrich for nuclear proteins?
Hello Iwan
I have worked with proteins from cell extract.
I advice you use RIPA buffer for the lysis (with proteasa inhibitors), incubated on ice 30 min and passed through a 20G gauge needle to facilitate cell breaking-up. After, centrifuge for 10000 rpm for 10 min and 4ºC.
Supernatant contains cytoplasmic proteins.
Pellet contains nuclear proteins
Best Regard
To detect a nuclear protein, you have two ways : first, total cell lysis, but you need choose the good one to disrupt the plasmic membrane and the nuclear membrane. Your protocol is this one. We need to know which total cell lysis you use ?
Second, you may purified the nucleus and after performed an protein extraction. You may need more samples to perform your purification but the protein lysat is less complex and the identification of your protein more easy.
Last, you may performed a DNA hydrolysis using DNase or ultrasonic treatment in order to dissociate DNA-protein complex. Often, DNA protein complex precipitate with insoluble proteins.
Hi Iwan,
what is the composition of your lysis buffer for nuclear extraction?
For nuclear proteins you can use the protocol from Mendez, J., and B. Stillman. 2000. In the case I just want to collect nuclear proteins I lyse the nuclei (fraction P1) in SDS-containing buffer. This can be sonicated or boiled for 10 minutes at 99 degrees and vortexed vigorously.
Hello Iwan
I have worked with proteins from cell extract.
I advice you use RIPA buffer for the lysis (with proteasa inhibitors), incubated on ice 30 min and passed through a 20G gauge needle to facilitate cell breaking-up. After, centrifuge for 10000 rpm for 10 min and 4ºC.
Supernatant contains cytoplasmic proteins.
Pellet contains nuclear proteins
Best Regard
Dear Iwan, how do you solubilise your pellet, containing the nuclear proteins? I also perform the protocol you mention, but I have troubles with resuspending my pellet after centrifugation.
Hi Iwan,
Make sure that you efficiently extract nuclear proteins during your sample preparation. Some lysis buffers (e.g. based on Triton X-100) may not extract nuclear proteins. I agree with the above try using RIPA buffer, the article below might be helpful as well: https://www.ptglab.com/support/protocols/. I would also highly recommend using nuclear loading control antibodies, such as lamin B1 or PCNA: https://www.ptglab.com/news/blog/loading-control-antibodies-for-western-blotting/. Alternatively, you may consider performing cell fractionation.
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