I would think thing here is that this protein actually associates to
those lipid nanodiscs...(around the "disc") and Na cholate CMC is
around 10 mM.
so, yes you can solubilise proteins that bind lipids, the question is
does this protein bind lipids or not? or is it just scrambled or
whatever, doesnt like to be overexpressed etc??? or sticky because it
is part of a larger complex naturally and not stable alone, for
instance. which i wouldn't know of course.
regards,
Tommi
On Apr 20, 2011, at 1:08 AM, Arthur Glasfeld wrote:
> I recently followed a protocol from Stephen Sligar's lab for the
> purification of his "nanodisc" protein, which has strong hydrophobic
> character as it associates with phospholipids. His protocol
> includes washes with 1% Triton X-100 and then with 50 mM cholate
> (both at pH 8 in the presence of 300 mM NaCl). Worked great, and I
> saw stuff coming off the column in both washes. The reference is:
>
> Bayburt et al. (2002) Nano Letters, vol. 2, pp 853-856.
> http://pubs.acs.org/doi/abs/10.1021/nl025623k
>
> Good luck,
> Arthur
>
> Arthur Glasfeld
> Department of Chemistry
> Reed College
> 3203 SE Woodstock Blvd.
> Portland, OR 97202
> USA
>
>
>
>
>
> On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote:
>
>> Dear colleagues
>>
>> We are working on a large bacterial protein (featuring a large
>> number of repeats) that appears to copurify with a lot of other
>> proteins after Ni-affinity chromatography and gel-filtration. We
>> have tried adjusting the ionic strength of these runs and have gone
>> to as high as 5M NaCl but only saw marginal improvements. It
>> appears that the protein likes to stick to a lot of stuff, and in
>> fact the number of repeats in a given construct appears to
>> correlate with the extent of contaminants in our purification
>> steps. We have admittedly never seen anything like this among the
>> so many different, and often challenging, proteins, we have worked
>> on in our group over the last few years.
>>
>> We are now thinking of trying detergents in the buffers (at non-
>> micellar concentrations), in conjunction with playing a bit with
>> the pH to see if such an approach provides a 'stripping' effect.
>> Interestingly, the protein has a calculated pI of 3.5 !
>>
>> As the options for handling this protein are indeed quite numerous,
>> we would be grateful for any additional input and possible tips/
>> tricks.
>>
>> I will prompty post a summary of the thread.
>>
>> Best regards
>> Savvas et al.
>>
>>
>> ----
>> Savvas Savvides
>> Unit for Structural Biology @ L-ProBE
>> Ghent University
>> K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
>> Tel/SMS/texting +32 (0)472 928 519
>> Skype: savvas.savvides_skype
>> http://www.LProBE.ugent.be/xray.html
>>
>>
>>
>>
>>
>>
>>
>
Tommi Kajander, Ph.D.
Structural Biology and Biophysics
Institute of Biotechnology
University of Helsinki
Viikinkaari 1
(P.O. Box 65)
00014 Helsinki
Finland
p. +358-9-19158903
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