Newbie question: Stability of protein lystates - Freeze/thaw cycles affect phosphate groups? (Aug/11/2009 )
Hi all,
I have some newbie questions regarding the stability of protein lysates. 
1. As far as I know, protein inhibitors have to be added fresh to the lysis buffer because they don't store well (?). After cell lysis, I froze my protein lysates at -80C. However, I repeatedly freeze/thawed my samples to do several SDS-PAGEs. Would the inhibitors lose their inhibitory activity, resulting in enzyme activity once thawed? As I am doing western blot of phospho-proteins, I am especially worried with phosphatases knocking off my phosphate groups. Surely I don't need to add another round of protein inhibitors every time I thaw them?! 
2. More generally, how should I store protein solutions? I've read that BSA solutions must be made fresh before use. Does this mean I can't store e.g. PBST+BSA or TBST+BSA (blocking buffers) solutions at room temperature? If I do make a stock, how does freeze-thaw cycles affect general protein solutions?
Thanks very much!!!! 
dc984 on Aug 11 2009, 09:22 PM said:
I have some newbie questions regarding the stability of protein lysates.
1. As far as I know, protein inhibitors have to be added fresh to the lysis buffer because they don't store well (?). After cell lysis, I froze my protein lysates at -80C. However, I repeatedly freeze/thawed my samples to do several SDS-PAGEs. Would the inhibitors lose their inhibitory activity, resulting in enzyme activity once thawed? As I am doing western blot of phospho-proteins, I am especially worried with phosphatases knocking off my phosphate groups. Surely I don't need to add another round of protein inhibitors every time I thaw them?!
2. More generally, how should I store protein solutions? I've read that BSA solutions must be made fresh before use. Does this mean I can't store e.g. PBST+BSA or TBST+BSA (blocking buffers) solutions at room temperature? If I do make a stock, how does freeze-thaw cycles affect general protein solutions?
Thanks very much!!!!
Hi DC,
1. Yes, freeze-thaw cycles can compromise protein samples. It would be better to store your lysates as aliquots, and defrost as-needed. This way you can minimize the number of freeze-thaw cycles you subject your samples to. The proteinase inhibitors will be OK as long as they are frozen, but if you plan on refreezing leftovers, maybe adding some fresh would be a good idea. (I don't do this b/c I make aliquots in the amount I need, with the P.I.s in them. The minimal surplus gets discarded.) Just remember to note the addition somewhere, in case you need specific concentrations.
2. You can store TPBS and TTBS + BSA in the fridge. In our lab, TBS and PBS are used by lots of people, so we make up large batches, aliquot into 500 ml bottles, autoclave, and store until used. I add the BSA when I run my WBs, and keep leftover solution, if any, at 4C. I've used TTBS+ BSA a month after I made it and it worked fine.
1. Concerning question one, you need to add protease inhibitors during the cell lysis because when a cell is lysed, all the proteases in the cell are released into the supernatant. Usually these proteases are contained in specific compartments (such as lysosomes). When the cell is lysed, they are released and can (and will!) start breaking down all the proteins in a sample. Most of the inhibitors in commercial preps are irreversible so there is no need to add new inhibitors every time the cell lysate is thawed.
What repeated freeze-thaw cycles will do is cause protein damage. THis is due to the ice crystal formation during freezing that will rip proteins apart. This will also cause a loss of the phosphate groups. Generally, when looking at phosphorylation, your best signal will come with fresh lysates. Some phospho proteins have to be analyzed fresh (such as phospho-AKT) while others (such as phospho-ERK) can handle one or two freeze cycles.
If you are going to freeze your lysates, add the running buffer to the lysates and divide into single use aliquots. Store at -80.
The only real problem with storing BSA solutions in the fridge is contamination problems. As long as you don't see contamination (which is really obvious!) you are fine. Don't add azide to these solutions to extend the life as azide will kill HRP.