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how to extract proteins from HEK293


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#1 eyes

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Posted 24 February 2011 - 02:24 AM

Hi I want to extract as much as possible proteins from transfected HEK293 cells. I indeed want to detect by wb a cytoplasmatic protein that should be very low expressed.
I seeded 2X106 in a 100 mm dishes and transfected with 10 ug DNA.
Could you please suggest me a protocol. It is not clear to me if it is better to detach cells with trypsin before the lysis, or it is better to lyse them directly with the loading buffer. Somebody then sonicate while other load directly the gel. I have seen that people for different cell lines uses different methods but I need something that works well for 293 and that allow me to understand if the protein I'm looking for is effectivelly expressed.
Thanks for help

#2 knuf

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Posted 25 February 2011 - 10:39 AM

Everyone has their own methods, but I think 293 cells should be pretty easy. What I do for them is put the plate on ice, aspirate out old media, rinse once in cold pbs (and aspirate out), then use a cell scraper with some volume of cold pbs (1 ml for a 60 mm plate)and scrape the cells off the plate. I then move the cells and pbs to a 1.7 ml tube and spin them down at 14,000 rpm for 45s, remove supernatant and resuspend them in 3 volumes of lysis buffer (25 mM Tris pH 7.4, 0.5% NP-40, 150 mM NaCl, and protease/phosphatase inhibitors). I then subject the cells to 3 cycles of freeze/thaw in a bath of dry ice and alcohol. I then remove the nuclei by centrifugation at 1000xg for 5 min at 4 deg C, and remove the lysates (this is your cytoplasmic protein)--you can measure the total concentration by bradford or whatever and add your loading buffer accordingly. For me, this is the most efficient way of doing multiple plates because you can freeze/thaw a lot of tubes at a time rather than sonicating or homogenizing one by one; however it is probably not the most efficient way of getting max amount of protein.

#3 eyes

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Posted 28 February 2011 - 08:46 AM

Everyone has their own methods, but I think 293 cells should be pretty easy. What I do for them is put the plate on ice, aspirate out old media, rinse once in cold pbs (and aspirate out), then use a cell scraper with some volume of cold pbs (1 ml for a 60 mm plate)and scrape the cells off the plate. I then move the cells and pbs to a 1.7 ml tube and spin them down at 14,000 rpm for 45s, remove supernatant and resuspend them in 3 volumes of lysis buffer (25 mM Tris pH 7.4, 0.5% NP-40, 150 mM NaCl, and protease/phosphatase inhibitors). I then subject the cells to 3 cycles of freeze/thaw in a bath of dry ice and alcohol. I then remove the nuclei by centrifugation at 1000xg for 5 min at 4 deg C, and remove the lysates (this is your cytoplasmic protein)--you can measure the total concentration by bradford or whatever and add your loading buffer accordingly. For me, this is the most efficient way of doing multiple plates because you can freeze/thaw a lot of tubes at a time rather than sonicating or homogenizing one by one; however it is probably not the most efficient way of getting max amount of protein.


Thank you kfunk106 for your replay.
I agree with you that probably everybody has its own method and I will habe mine in the future but starting with somebodyes' else experience is for me a better starting point.
I have a couple of question. When you say ".....resuspend them in 3 volumes of lysis buffer" did you mean three times the volume of cold pbs used for scraping?
And when you say "......I then remove the nuclei by centrifugation at 1000xg for 5 min at 4 deg C, and remove the lysates (this is your cytoplasmic protein)" as lysate did you mean the supernatant?
Thanks again
Gianluca

#4 knuf

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Posted 01 March 2011 - 10:45 AM

Hi Gianluca, to answer your quesetoins,

When you say ".....resuspend them in 3 volumes of lysis buffer" did you mean three times the volume of cold pbs used for scraping?


No...I mean to resuspend them in 3 volumes of lysis buffer, which is composed of 25 mM Tris pH 7.4, 0.5% NP-40, 150 mM NaCl, and protease/phosphatase inhibitors

And when you say "......I then remove the nuclei by centrifugation at 1000xg for 5 min at 4 deg C, and remove the lysates (this is your cytoplasmic protein)" as lysate did you mean the supernatant?


Yes, lysate=supernatant=cytoplasmic protein. pellet=nuclei.




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