Aggregation in dilute samples

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wunfuntin
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Aggregation in dilute samples

#1 Post by wunfuntin » 2014.06.05 17:00

We have some samples that it seems that in the dilute samples (0.5mg/ml, 1mg/ml) that there is more aggregation observed in the scattering profile than in the higher concentrations (2mg/ml, 4mg/ml). Has anyone seen this before? Is it possible that total amount of aggregation is the same for each sample, but that the higher intensity derived from the more concentrated samples are some how masking the presence of aggregation? I tend to think that this would not be case but figured I would throw it out there.

For technical reference, these samples were sized and then pooled from the peak and placed into an Amicon concentrator. As the samples were concentrated, we took off aliquots of the sample when they reached the above mentioned concentrations (so as to alleviate any dilution effects). We also observed this in 4 different buffers (so unless we try a battery of buffers/additives, I am not sure of what else to do).

Thank you for your help!

Ryan

Martin_NP
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Re: Aggregation in dilute samples

#2 Post by Martin_NP » 2014.06.12 16:16

in my experience, samples with lower protein concentrations has higher probability of exhibiting radiation damage than high protein concentration samples. Do you have several frames from each data acquisition? if so, compare the frames and look for any development.

wunfuntin
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Re: Aggregation in dilute samples

#3 Post by wunfuntin » 2014.06.13 14:52

Hi Martin_NP,

Thank you for your response.

I guess it could be radiation damage but the canonical indication of radiation damage is not present (i.e. discrepancies/inflection points in the multiple frames throughout the numerous exposures 30exposures/sample at each concentration). Therefore, I doubt that radiation damage is the cause. Plus the samples have 2% glycerol in them so I would expect most of the radiation damage to be minimal. The aggregation I am seeing is pretty severe.

Ryan

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