3 replies to this topic

[zatler]

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

[Dr Teeth]

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

[yobou]

I always add commercial phosphatase inhibitor cocktail to lysis buffer.

[laurequillo]

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...)