2 replies to this topic

[tiki]

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...!!!

[eldon]

tiki, on Jul 29 2009, 09:52 AM, said:

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...!!!

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.

[tiki]

eldon, on Jul 29 2009, 05:32 PM, said:

tiki, on Jul 29 2009, 09:52 AM, said:

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...!!!

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.

I´m 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 don´t add it fresh. I will try reboiling ....
thanks...thanks for your advices

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