Fluorescence polarization buffer

D.K. no.email at thanks.to.spam.net
Fri May 2 18:58:00 EST 2003


In article <Eezsa.42$v5.9168 at news.uchicago.edu>, "EK" <REMOVEem at NOSPAMunforgettable.com> wrote:
>
>"D.K." <no.email at thanks.to.spam.net> wrote in message
>news:b8u0l4$kpp$1 at news.doit.wisc.edu...
>> In article <PaVra.10$v5.1032 at news.uchicago.edu>, "EK"
><REMOVEem at NOSPAMunforgettable.com> wrote:
>> >I am trying to find conditions to stabilize my protein in dilute form for
>FP
>> >and other assays that require long times of exposure at room temperature.
>> >Could someone hint on what agents could I try?
>> >One company sells a FP kit and claims the buffer they provide contains
>> >glycerol, protein "stabilizing agents", and agents to reduce high
>> >backgrounds due to unspecific binding (they certainly not disclosing
>their
>> >buffer recipes). I know that the protein stabilizing agents are DTT and
>> >protease inhibitors, but it seems like they added something else to the
>> >buffer. Any ideas what could that be? As for background reduction, the
>well
>> >known additives are BSA and BGG (gamma globulin). However, the company
>says
>> >those two are not present in the buffer. What else is know to reduce
>> >backgrounds and at the same time stabilize unstable proteins in dilute
>> >solutions?
>> >
>> >Any ideas/comments/suggestions will be much appreciated.
>>
>> Ethylene glycol frequently works better than glycerol.
>>
>> Betaine would be another thing to try - it increases melting
>> temperature of proteins (e.g. stabilizes against spontaneous
>> denaturation) and, being a zwitterion, decreases non-specific
>> interactions.
>>
>> There should be nothing wrong with BSA and if I were you, I'd
>> add it. Probably the best thing you can do to keep very dilute
>> protein stable.
>>
>> DK
>
>Thanks. They (company) say the buffer is a "product of years of research",
>and BSA or BGG are not in it, meaning, they probably used some of the other
>stuff you and Kyle mentioned. I recall something like up to 30% EG, and ~1M
>betaine. Is that correct? 

Yeah. 15-20% EG and 0.5-1.0 M betaine is where I'd start. Whatever 
the company says, I'd still throw in 0.1 mg/ml BSA. 

>Any tips on qualitative tests for buffer
>composition? Of course, I'll do SDS-PAGE to check for the proteins/peptides,
>check absorption spectra, see if refraction index is high (EG, high salts).
>Anything else comes to mind? I am not sure I'll go far enough to do
>ninhydrine test, but I might :-).

I have no idea. Last time I dealt with fluorescence [de]polarization 
was during my undergrad study - you know how long ago it was :-) 
I suppose if you have an activity test for your protein, you can 
just test it. Other than that I am afraid there is no other way but
do the real thing (your EP study) and see what works best. 

Dima



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