Forums
Bruker Media
Community Media
Bruker AFM Probes
SPM Digest
Application Notes
NanoTheater
Website
中文
Brochures & Datasheets
Publications
Probes Catalog
Events
Manuals & Documentation
Presentations
Guide to AFM Modes
News
Journal Club
Webinars & Video
Nanovations
Other
Hi,
does the drying of the already adsorbed biological sample (like proteins) before immersing in liquid make any difference from adsorbed sample in liquid without intermediate drying step? With term "dried sample" I mean that it was prepared with procedure for imaging in air. I am asking this because I am concerned with contamination of tip during adsorption process when cantilever is already mounted in fluid cell and we have to wait that protein is immobilized on mica during adsorption.
Can anyone provide his experience/procedure for preparation of sample preparation and mounting of the cantilever in the cell when the imaging of adsorbed proteins on mica at stagnant condition is required (or even for direct observation of adsorption dynamics)?
I am using Nanoscope IIIa in standard fluid cell and I am using mica as substrate for protein sample in buffer
Thanks,
Dejan
Hi Dejan,
I believe it has some importance although I never saw anything related to this in the literature. It's obvious for large structure like cells like the drying step dramatically affect the cell structure. But I remember an experience on antibodies (anti-lysosyme) during my Postdoc: when drying after adsorption in liquid, and imaging in fluid, the antibodies never exhibit the same structure compared to a situation where antibodies where constantly (adsorption and imaging) kept under liquid environment. This is especially true for a higher density of molecules since the degree of compaction is strongly affected by the hydration state. I am not sure the comparison is very relevant but just think about lipid bilayers: if you dry them out and re-hydrate them, you won't have bilayers but an alternance of mono- and trilayers.
Alex.
Thanks Alex
Yes, I also think that you have right. I was asking that, because I saw in some literature (imaging of DNA from Lyubchenko) this approach.
Do you have any advice with cantilever and fluid cell assembling to reduce any possibility of tip contamination? Now, I deposit the droplet of sample on freshly cleaved mica and assemble the fluid cell with attached cantilever and O-ring on the scanner head. This I leave for about 15-20 minutes to adsorbed. But, I have problems with tip contamination. If I try with increasing the tip-sample distance to the highest possible the tip is still in contact with droplet of sample.
Dejan,
Something that works quite nice is a hydrophobic coating on the probe. You might slightly compromise on the resolution but definitely benefit contamination wise!
Did you ever try this?
No, I've never tried this, but sounds interesting. The only concern I have is that I am wondering if this could have influence on sample because of different tip-sample interactions (unfolding of protein due to hydrophobic tip contact?).
Could you provide more information/details about this?
Thanks for this suggestion,
You are absolutely right: this approach is interesting if you image cells and if you want to prevent the cell debris to contaminate the probe (and same for lipid bilayers). Regarding proteins, I am not sure how a hydrophobic coating would affect the imaging and the proteins themselves. I would say that this depends on the imaging technique. If the probe remains in contact with the proteins for a long time, the vicinity of it might affect the protein structure (folding or unfolding) but in tapping mode or peak force tapping, the interaction time is too short to impact the protein structure. At least this is my feeling because I have no precise paper or document to refer to.
Best,
I am doing in tapping, so this should not has the great impact.
I will try to estimate the possible tip contamination on uncoated tips. Maybe is the best way to characterize tip and then measure the adsorbed protein in fluid with the same tip to estimate possible contamination. And then hope that the contamination is insignificant. In the case of no success it may be better to try with hydrophobic coating. Which kind of coating do you use?
I suggest OTS with 10 or 12 carbons. If you start from a bare Si probe, that's probably the simplest way. If you start from a gold-coated coated probe, you can use alkanethiols with the same number of carbons.
Good luck,