I am looking for a rather simple protein aggregation assay that I can use in class to teach students about protein damage, but that is also fairly reliable. Everything I have seen for the lab requires equipment not available in the classroom and perhaps I will have to resort to that but I am wondering what is out there.
If you want cool and simple, consider this. Light scattering. You can for example shine a red laser pointer through a protein solution, then you aggregate/ppt it and you will see the laser path in the solution. Similar to dust particles in a room. You might even use a different laser (green) scattering strongly depends on the wavelength for a given particle size. This could be quite visual and simple... Now if I just can get a NICE blue laser
Use BSA some well know and available protein for model! Zn or some other ions may help your protein to aggregate quickly.
If you have access to a UV spectrometer you could perform some sedimentation assays. Simply follow the 214 nm absorbance (or 280 nm if you have enough signal) and scan the supernatants (after centrifuging timed samples) to follow the aggregation - get the students to express the data as %protein in solution. Addition of Zn2+ as Ananda suggested will help to speed up the process, as will incubating at higher temperatures or changing the pH. You could also get the students to compare total protein vs supernatant by spot-blotting (dot-blotting) - all you need is some nitrocellulose/PVDF and a staining agent such as ponceau or coomassie.
Try heating BSA, BSA when heated to 50°C or above, albumin quite rapidly forms hydrophobic aggregates which do not revert to monomers upon cooling. Even aggregation of BSA happens at lower temperatures, but it occurs relatively slower in rates.
If all you need just show protein aggregation - use an egg on a hot plate. It's the best simple visualisation I've ever seen :)
Circular Dichroism is fairly easy to do but not suitable in a classroom setting.
Amyloid peptides monomeric in in HFIP will aggregate within 6 hours by adding water or organic solvent, but you would need Circular Dichroism.
Staining with Thioflavin T, that stains only protein aggregates may work but I am not certain in a classroom setting.
For a classroom setting if you want to show irreversable protein denaturation, use egg white and heated (just like boiling an egg). The denaturation is totally irreversible. Natural egg white is transparent or translucent. After denaturation, it will form a solid white mass. You could instead denature it with an organic solvent (e.g. acetone).
I don't know if this is helpful...but is always easy for the students to relay to.
Casein aggregation after addition of a few drops of acetate buffer to get pH aprox.pI (around 4.2, maximal turbidity) is also very easy and suitable for a classroom demonstration. Is that what you are looking for?. Cheers!
What's heavily been used at least for some amyloid proteins is turbidity (that is, absorbance at something like 405 nm) in a buffered solution. Tried it myself, it works if the proteins you study do aggregate and, being mesasured at 405 nm, its visible, meaning that in some cases you can watch your samples aggregate with the naked eye. Did so on 96 well microplates.
Thanks for the info. I am in fact looking for both, a cool demonstration I can show undergraduates but also a working assay I can have graduate student perform on say Drosophila, after exposing them to heat.
If you want cool and simple, consider this. Light scattering. You can for example shine a red laser pointer through a protein solution, then you aggregate/ppt it and you will see the laser path in the solution. Similar to dust particles in a room. You might even use a different laser (green) scattering strongly depends on the wavelength for a given particle size. This could be quite visual and simple... Now if I just can get a NICE blue laser
Differential centrifugation works for some (soluble protein stays in supernatant, aggregate pellets), also filtration of lysate/protein sample through high-molecular-weight cutoff filter (e.g., Microcon 100) works. If the aggregate is "amyloid"-like, it should be SDS-resistant, and you can add 1% SDS to your lysate/protein sample prior to filtration to make everything else denature. Finally, there is a form of agarose gel electrophoresis (SDDAGE) that separates protein aggregates.
To Michael McMurray:
Did you mean that SDDAGE separates protein aggregates SDS-resistant in SDS-PAGE? Could you please give more details about conditions of SDDAGE or some reference? Thank you!
Sure, Francisco: See "Analysis of amyloid aggregates using agarose gel electrophoresis." Sviatoslav N Bagriantsev, Vitaly V Kushnirov, Susan W Liebman
Methods in enzymology. 2006 Oct; 412:33-48. I can send you the PDF if you don't have access to the journal.
OK, I think that I have access to that collection in my University library. Thanks a lot!
Michael Mcmurray, Could you please send me the attachment. I am very interested in learning about SDS-resistant proteins? Thanks a lot.
Perhaps you can consider Split Green Fluorescent Protein (sGFP) technology. This is a novel technique whereby aggregated proteins will obscure the sGFP tag and prevent complementation with the probe. Successful disaggregation can be detected and quantified with the probe when the tag becomes available once more. Proteins that have become insoluble will not be available to the soluble probe either and will thus complement in a limited manner and attenuate fluorescence. Further, this technique provides extremely high specificity and sensitivity while virtually eliminating non-specific signaling and background. Please visit http://www.sandiabio.com.
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