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Phospatase inhibitors needed for LDS Buffer?


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#1 zatler

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Posted 05 March 2009 - 02:13 PM

Hi,

I am trying to look at the phosphorylation of the gene c-Met. I do not have a lot of experience with Westerns, however another lab is helping me out and they tell me to lyse cells in laemmli sample buffer and it stops all reactions including phophatases. However I have read in most cases people use RIPA buffers supplemented with protease and phophatase inhbitors.

Is it true that LDS sample buffer will stop all reactions including phosphatases which might cause the dephosphorylation of the gene I am trying to look at, C-met. Thanks

#2 Dr Teeth

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Posted 06 March 2009 - 08:51 AM

You should add external phosphatase inhibitors during the lysis to prevent dephosphorylation before reaching addition of LDS buffer.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
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#3 yobou

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Posted 13 March 2009 - 09:11 PM

I always add commercial phosphatase inhibitor cocktail to lysis buffer.

#4 laurequillo

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Posted 25 September 2009 - 05:12 AM

If you use a "normal" lysis buffer you shoul add the inhibitors, but with laemli buffer you dont need them. You just have to lysis the cells directly in 1x lysis buffer, boil for 2 minutes, sonicate 2x15 sec, boil 5 minutes, and load the samples. In this way you can see most post-transcriptional modifications (phosphrylation, ubiquitination, sumoylation...)
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