I am having difficulties to image protein fibrils using AFM. What is the ideal concentration of amyloid fibrils that I should use? Also how long does it need to sit on the mica surface before I dry it off?
I'm quoting this from my labmate's paper:
Role of Small Oligomers on the Amyloidogenic Aggregation Free-Energy Landscape
doi:10.1016/j.jmb.2009.10.019
"Each day, after inversion of the tube once, 20 μl of the incubated solution was aliquoted for AFM imaging on modified mica. To modify the mica surface, we applied 20 μl of 0.1% (vol/vol) APTES (aminopropyltetratheoxysilane, catalog no. 151081000; Acros) evenly on a freshly cleaved 9.9-mm-diameter muscovite mica disk (product no. 50; Ted Pella) and allowed it to react for 10 min. Unreacted APTES was rinsed away with 15 ml of Millipore water. The surface was dried with HPLC-grade compressed nitrogen gas. The incubated sample was applied evenly on this freshly prepared surface and allowed to adsorb for 3 min. Unbound species were rinsed away with Millipore water. Residual water was blown away with nitrogen gas. The sample was imaged by a MultiMode Scanning Probe Microscope with a Nanoscope IIIa controller (Veeco), with a tapping-mode etched silicon probe (TESP; Veeco) in tapping mode in air. The scan speed was 1 Hz, with an image size of 512 × 512 pixels. Samples were stored in disk carriers when further imaging was required."
best of luck!
I prefer to try different concentrations protein fibrils (in the context of initial monomeric concentration). I got good results with fibrils diluted to 0.2-0.5 mg/ml concentrations. I apply a 10 ul fibril sample drop on mica surface and let it sit for 15-20 minutes, followed by washing with MilliQ water. If your fibrils are formed at acidic pH, it is advisable to acidify water before washing. The washing should be done gently by adding water over the surface in dropwise manner using 1 ml pipette (dont squirt water jet on mica surface). You must carry out washing in order to remove buffer salts and other solutes from mica surface.
The mica should be dried by placing it under vacuum desiccator or by placing it under gentle stream of nitrogen. I got satisfactory results by letting it dry for few hours or overnight under normal laboratory conditions.
What sort of difficulties are you having? If you are not seeing any fibrils on the mica, there could be a few things to consider. The other answers cover the preparation but I'd like to add that some fibrils are quite "sticky" and tubes need to be vortexed vigorously, sometimes even scraped off the side of the tubes with the pipette tip. If that is the case, there could be a large number of fibrils not in suspension so your concentration could be less than you believe.
It is also necessary to take into account the possibility of interaction of amyloid fibrils with cations or anions of mica with the formation of non-covalent bonds.
You might want to check the AFM experimental section of this paper.
https://www.nature.com/articles/srep40787
We have used AFM to image amyloid-beta fibrils on mica.
- Badges
- Science method