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help...my RIPA does not working..!!!


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#1 tiki

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Posted 29 July 2009 - 08:52 AM

my RIPA is aliquoted and stored at -20 d
when I used it did not work well how before it did and I got poor protein content in my samples (from culture cells), indeed I were surprised because my samples became like a gel and was so difficult to take a bit (with micro) and of course to do western blot and measure protein.
any help will be welcome...
sory for my english...!!! :(

#2 eldon

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Posted 29 July 2009 - 12:32 PM

my RIPA is aliquoted and stored at -20 d
when I used it did not work well how before it did and I got poor protein content in my samples (from culture cells), indeed I were surprised because my samples became like a gel and was so difficult to take a bit (with micro) and of course to do western blot and measure protein.
any help will be welcome...
sory for my english...!!! :lol:


how much ripa per sample? make sure you add protease inhibitors.

60ul-100uL is sufficient for a confluent 6-well plate...not all 6 wells...100ul per well of cells. scale it up for 10cm dishes.

rock the samples in a cold room for an hour and then briefly sonicate. spin the samples for 10min at 14k rpm to remove the insoluble material. transfer the soup to a fresh tube and store frozen (-20C). alternatively, quantify the sample right away and then do your western. i don't add sample buffer to my stock protein...i take an aliquot equal to 20ug, and add sample buffer to that, then boil 5 min.

repeated freeze/thaw will ppt the protein or make it gelatinous. try reboiling.

#3 tiki

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Posted 29 July 2009 - 02:42 PM

my RIPA is aliquoted and stored at -20 d
when I used it did not work well how before it did and I got poor protein content in my samples (from culture cells), indeed I were surprised because my samples became like a gel and was so difficult to take a bit (with micro) and of course to do western blot and measure protein.
any help will be welcome...
sory for my english...!!! :lol:


how much ripa per sample? make sure you add protease inhibitors.

60ul-100uL is sufficient for a confluent 6-well plate...not all 6 wells...100ul per well of cells. scale it up for 10cm dishes.

rock the samples in a cold room for an hour and then briefly sonicate. spin the samples for 10min at 14k rpm to remove the insoluble material. transfer the soup to a fresh tube and store frozen (-20C). alternatively, quantify the sample right away and then do your western. i don't add sample buffer to my stock protein...i take an aliquot equal to 20ug, and add sample buffer to that, then boil 5 min.

repeated freeze/thaw will ppt the protein or make it gelatinous. try reboiling.


Iīm new in bio-forum and sometime I touch incorrect icons....I have to learn more...
Thank ELDON for replying!
I usually use 500uL for a confluent 25 cm flask (what do you think ?) and do the procedure very close to you...but I will give attention step by step. The buffer have protease inhibitors, I donīt add it fresh. I will try reboiling ....
thanks...thanks for your advices :lol:

Edited by tiki, 29 July 2009 - 02:45 PM.





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