[RASMB] aggregates data analysis

[Joris Beld]

Dear all,
Sorry for not replying earlier. First a few remarks:
- the 'take off' point goes down the cell at higher speeds
- 30krpm was too fast but since the monomeric species is only 4kDa and 
we had no idea about the aggregating state we chose as a first speed 30krpm
- Thanks to Allen Minton for the reprint! This looks similar to what we 
observe.
- The ionic strength of the solution is low (10mM) and it's a 10% 
methanol/water solution
- ultrascan: I used the single-ideal-species model to fit the data as 
well as the fixed MW model (from 10k-1MDa) and both do not fit well 
since the curvature is very non-ideal

I have attached an example of a scan at equilibrium (two days) at 
10krpm. The data is not great. Data has been obtained at 260nm, 20C, 
120ul of liquid column and 30ul of FC43.

For me it seems like it could be two things:
- either this peptide/protein aggregates heterogeneously and forms a 
whole bunch of different sized aggregates (which would make sense with 
-for example- our CD measurements and it would make sense with the 
designed characteristics of this peptide/protein)
- or the conditions are so non-ideal with the low salt concentration and 
the methanol present that this influences the behavior too strongly.
I'm running now a SV run to see whether this behavior is also seen in 
this experiment.
Thanks a lot for the help.
Best,

Joris



Leech, AP wrote:
> Hello Joris,
>
> I would expect the "take off point" to move down the cell at higher
> speed. Does this happen? Do you have scans for the approach to
> equilibrium to see how this distribution develops?
>
> For 280 kDa and 30000 rpm I would also expect everything to be pelleted
> at the bottom. Do you have an expected Mw for the sample?
>
> It might be useful to post a plot of the data. If it is not some sort
> of artefact then maybe a velocity experiment would be informative.
>
> Best regards,
>
> Andrew
>
>
> Beld, Joris wrote:
>> Dear all,
>>
>> I'm spinning a sample which shows -to me- surprising behavior. The 
>> raw data of a normal sedimentation equilibrium run of a protein looks 
>> always like a half-parabolic curve of the radius versus absorbance 
>> (x,y).
>> However, this particular sample (protein) shows a different behavior 
>> in that the raw data look like a straight line taking off halfway the 
>> liquid column (absorbance almost zero) in the sector to the bottom of 
>> the cell (absorbance 0.7). Same behavior is reproducible at three 
>> different concentrations and three different rotational speeds 
>> (10,20,30 krpm).
>> How should I interpret these data? Ultrascan does not really 'like' 
>> this data as input (although fitting is possible, the residuals look 
>> very bad, the fitted Mw -to a single species- is around 280kDa).
>> Is this how a heterogeneous aggregating sample should look like?
>> Thanks in advance.
>>
>> Best wishes,
>>
>> Joris Beld
>> ETH Zürich
>> Switzerland
>> _______________________________________________
>> RASMB mailing list
>> RASMB at rasmb.bbri.org
>> http://rasmb.bbri.org/mailman/listinfo/rasmb
>>
>


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