Cells not lysing efficiently in hypotonic buffer. Help!

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Cells not lysing efficiently in hypotonic buffer. Help!

Postby jwb » Jan 03 2017 3:45 pm

I am fractionating some mammalian cells (a tumour cell line) to obtain nucleus/debris pellet (800g), crude mitochondrial pellet (10000g), microsomal pellet (100000g) and the post-microsomal supernatant. However, I find that the hypotonic lysis buffer I use probably does not lyse the cells effectively (in fact, the lysis seems really poor), in spite of me incubating harvested cells in it for half an hour, followed by passing several times through a 26-gauge needle. This I know, because most of my protein is found post-lysis in the 800g pellet (probably unlysed cells). My protein is not found in the nucleus inside cells.

The lysis buffer I use is 20M Tris, 5mM MgCl2, 5mM CaCl2, 1mM EDTA, DTT, + protease inhibitors. What do I do to lyse cells effectively? I currently scrape cells with a rubber scraper. Will trypsinisation give better results? Some protocols call for Dounce homogenizers, but I currently don't have any. I have a Potter-Elvejhem homogenizer, though, but the volume of the homogenizer is a bit high (5 mL). Are Dounce homogenizers better than Potter-Elvejhem for hand-held lysis of cells? I can't find this information easily.

What is a foolproof method to ensure near-complete cell lysis, keeping nuclei and organelles intact? :( :cry:
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Re: Cells not lysing efficiently in hypotonic buffer. Help!

Postby relaxin » Jan 04 2017 3:20 pm

You can try 50 mM Tris HCl pH 8.5, 150 mM NaCl and 1% NP-40. NP-40 will not lyse the nuclear membrane.
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Re: Cells not lysing efficiently in hypotonic buffer. Help!

Postby jwb » Jan 04 2017 3:50 pm

Hi relaxin,

That is usually the buffer I use for quantitative western blots, but this time I am trying to fractionate without detergent because I believe the protein I am probing for loosely binds to membranes. NP-40/Triton extract it easily, hence I'm using no detergent while fractionating. My objective is to retain the protein on the membrane.
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Re: Cells not lysing efficiently in hypotonic buffer. Help!

Postby relaxin » Jan 05 2017 10:25 am

If you do not want to use detergent, then you have to use Dounce homogenizer. You can ask the other labs in your department and borrow one.
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Re: Cells not lysing efficiently in hypotonic buffer. Help!

Postby jwb » Jan 05 2017 3:54 pm

Yes, I guess that's what I eventually have to do. They're quite expensive, though, and need to be washed between each sample. Yesterday, I tried freeze-thawing after swelling in hypotonic buffer. Since I did it in an 1.7mL tube, it was quite fast and seemed to lyse cells well. I stained the lysate with Trypan Blue and saw that >95% cells were lysed. I will know when I do western blotting if the method has worked well.

Also, I trypsinized the cells this time instead off scraping them off the dish. I think I've realised that scraping is a lousy method and should never be preferred to trypsinization unless there's a specific reason not to trypsinize. Especially where detergents are not used.
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Re: Cells not lysing efficiently in hypotonic buffer. Help!

Postby relaxin » Jan 07 2017 1:05 pm

If freeze-thaw method works for you, then it will be fine. I recall some people pass the cell suspension through a narrow pore needle with a syringe, but I do not remember the gauge size of the needle. If you use trypsinization, be sure to add proteinase inhibitor cocktail during the cell lysis, or you may have degradation of your protein by trypsin.
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