Protein extraction from animal cells - (Jan/09/2008 )

Hi, how to extract protein from animal cells?
Anybody doing protein extraction in your lab or for ur experiment? Hope to get some guidance from u all...Thanks

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hi friend
in my lab, total proteins are extracted from aortas of rats for Western blot.

we use a homogenisation buffer includes (I dont remember concentrations, sorry )
TrisHCl 31.5mg, EGTA 7.6 mg,EDTA 18.6 mg. (final volume 10 ml and ph 7.5) store at 4+

After this, protease inhibitors are added (for 10 ml homogenisation buffer)
3.3 mg DDT,
5 mg aprotinin,
5 microliter leupeptine (from stock -1 mg leupeptine per 0,5 ml distiled water, store -80 ),
10 microliter pepstatin (from stock -1 mg pepstatin per 1 ml distiled water, store -80 ),
50 microliter PMSF (from stock -17.4 mg PMSF per 1 ml etanol, store -80 )
finally you will obtain homogenizastion solution

homogenate your animal tissue (w/v 1/1) using mechanical homogenizator
for example, add 1000 ml 100 mg tissue.

and then santrifuge at 13.000 rpm 30 minutes 4+
bye

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Hi friend,

Thanks for your protocol. I am actually dealing with cell culture work. My lab don't have homogenizator, any other methods available? Thanks.

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Try to freeze-thaw cells suspension three times with this buffer, or if you dont want to do the F-Z, add 0.1% triton X-100 to the buffer.

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0.1 % triton X-100 in PBS is enough to extract out proteins from the cells? No need to follow by freeze-thaw method?

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no need.

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homogenate your animal tissue (w/v 1/1) using mechanical homogenizator
for example, add 1000 ml 100 mg tissue.

The volume is ul, not ml, right?

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Ok...thanks for your suggestion.

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