1% boiling buffer for protein extractions - (Nov/13/2008 )
Hi, new to the site, and relatively new to doing western blots and protein extractions. I've googled this topic on the forums before, and I've found some answers that were helpful. This might be a stupid question, but I was given a protocol for protein extraction that called for lysing cell samples (2 million neuronal cells plated in each well of a 12 well plate) using 50uL of what is called a "boiling buffer". This boiling buffer contains 1% SDS in 1x PBS.
I was confused as to whether this means you boil the buffer before adding it to the sample? Every other protocol I've read and person I've talked to has mentioned that it is imperative to collect lysates and do all the work on ice. I can't imaging adding hot lysis buffer to the cells would be good? Wouldn't the heat destroy any protease inhibitor tablets I add to it? Is this just a misnomer?
Thanks for reading!
I was confused as to whether this means you boil the buffer before adding it to the sample? Every other protocol I've read and person I've talked to has mentioned that it is imperative to collect lysates and do all the work on ice. I can't imaging adding hot lysis buffer to the cells would be good? Wouldn't the heat destroy any protease inhibitor tablets I add to it? Is this just a misnomer?
Thanks for reading!
There are essentially two ways to extract proteins for westerns. One is to lyse the cells open using a mild buffer and some sort of shearing force such as sonication, vortexing, etc. This keeps the proteins intact and (hopefully) in their native confirmation. This is where you use protease inhibitors, etc. The other way is to put your cells directly into the loading buffer that will be used for the western (similar to the SDS buffer you mentioned) and to boil the samples. This denatures the proteins and makes them immediately ready for western blot. Using this method, the proteins will not be suitable for other experiments such as co-ip, enzymatic assays, etc.
Thank you.
However, if I put cells directly into the loading buffer (contains BME and bromophenol blue etc), I can't then be able to do a protein estimation, right?
Thank you.
However, if I put cells directly into the loading buffer (contains BME and bromophenol blue etc), I can't then be able to do a protein estimation, right?
that is correct.