How to bake membranes without detergent ? - (Jul/26/2006 )

Helloooo

I need some help... I need to break the cytoplasmatic membrane of PC12 cell... but I need a protocol without detergent.... I'm thinking in some kind of Freeze fracture or something like that
If someone can help me...

Thanks everybody

Kiklio

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You have several options.

A French press will mechanically break the membranes.

Try lysozyme (I think it's 4mg/ml but play around and see what works for your cells). Lysozyme enzymatically breaks the membrane.

You might be able to achieve cell breakage simply by changing the buffer. e.g. I bust open red blood cells by changing from serum to a weak phosphate buffer and centrifuging for a few minutes.

I'm not sure how robust your cells are but your aim is to bust open the cells without damaging whatever protein you want then to isolate.

Try all and choose the one that fits best. Also, do check the literature.

Hope that helps.

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You can freeze-thaw your cells in PBS: 10 sec in liquid nitrogen (or 2 min in dry ice) and 5 min at 37 degrees. Repeat 5 times

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Thanks all for the help
One more question..
After the freeze and thaw ... Make a centrifugation for separete the braking membrane ??
Thanks again

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Yes

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you can also lyse the cell using syringe (27G) in a hypotonic/isotonic lysis buffer. Starting with 3 strokes.
To make use nuclear content doesn't release, you can take out serveral ul cells and stain with trypan blue. Cell with intact plasma membrane will appear as white and nucleas will appear as blue (coz prypan blue can stain nucleas but cannot pass through plasma membrane)

Over ~80% of membrane lysis is acceptable.

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