I'm looking for alpha synuclein aggregates. I have a few questions about the technique.
Hi Kellie, I have done many protein filtrations, mostly with abeta and hamster prions. The thing is that you can buy filtration units with cutoff from 1KD to 100,000 KD.
Problem is that mis-folded proteins like synuclein, Huntington, prion, abeta do not form according to size. They are all over the place with respect to size.
Are you referring to a large aggregate filtration assay using microfiltration as opposed to ultrafiltration? Are the targeted aggregates actually fibrils larger than submicron particle sizes involving the use of SDS? If you're asking about size exclusion selectivity at 0.1 or 0.2 microns then there probably is not very much relative to tangential flow ultrafiltration. However, selectivity may not matter if you're interested in total particles only in the micron range and have a sensitive immuno-detection method requirng very small sample quantities to avoid polarized gel concentrate effects.
One of the issues you may encounter is protein aggregates binding to the membrane. I found significant binding of large protein assemblies with a range of high molecular weight oligomers and amyloid fibres from a range of proteins and peptides such as cystatin B and amyloid beta peptides. As aggregates increase in size you are increasing the likelihood and binding sites present on each particle/fibre. Try using buffers with 100-200 mM NaCl to shield electrostatic interactions if possible, but this may reduce the stability of your aggregates in the case of reversible aggregation. Perhaps the best method might be ultracentrifugation with density gradients.
Thank you Peter! Do you have any other suggestions or different assays that might be better?
It depends upon what you need to know regarding the alpha synuclein aggregates. If you want to characterise the aggregates you could try a filter immunoassay blot, whereby you filter your protein preparation through a filter which does not retain the monomeric protein. Then use an immunoassay with oligomer-specific antibodies etc. from groups like Charles Glabe et. al. e.g. antibody A11? and OC. Eric Wanker uses these assays a lot to excellent effect. This may indicate certain properties of your aggregate etc. and could be used as a crude method of quantification. You could try using assymetric flow fractionation (AFF) which has been used very successfully for prions. It all depends upon what you need to know.
Peter & everyone:
This is the protocol I was intending to use, any thoughts? tricks to simplify or important areas to be due diligent?
Samples for brain homogenates were taken from the frontal cortex (gyrus frontalis medialis) in DLB cases and from the substantia nigra in PD. They were homogenized in 9 vol (w/v) MSE buffer (10 mM MOPS/KOH pH 7.4, 0.3 M sucrose, and 1 mM EDTA) including protease inhibitors (1 mM PMSF, 0.2 mM TPCK, and 0.2 mM TLCK) with a glass/teflon homogenizer by 12 strokes.
Protein Aggregate Filtration Assay for α-synuclein Aggregates
After centrifuging brain homogenates at 16,000 g for 5 min, supernatants
supernatants were aspirated and saved for later analysis. The pellet fractions were subjected to digestion with DNase I (Fluka, Buchs, Switzerland) in 4% N-tetradecyl-N,N-dimethyl-3-amonio-1-propane sulfonate (SB14; Sigma) for 15 min at 37°C. Finally, pellet and supernatant fractions were adjusted to a final concentration of 5% SLS (Fluka).
For protein aggregate filtration, a commercially available slot blot device (Bio-Rad Laboratories, Munich, Germany) was used. Samples were filtered through a blocked nitrocellulose membrane (0.2 μm pore size, Schleicher & Schuell, Dassel, Germany). After filtration, each slot was washed with 0.1% SDS. α-synuclein aggregates were detected using the monoclonal antibody LB509 (1:10,000) with a secondary HRP-coupled goat-anti-mouse antibody and finally visualized by chemiluminescence. Densitometric quantification of PAF blot was performed with SigmaGel v1.0.
At 0.2 um the filters will only retain large aggregated material. You could reduce the membrane pore size to observe a range of sizes. Good luck!
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