Dear Amna,

I am using AFM for the study of protein-DNA interactions as well. Some thoughts concerning your questions are summarized below.

- Freshly cleaved mica is negatively charged when exposed to water, and in buffers with high ionic strength, an extensive accumulation of cations near the interface will take place. This in turn can, in principle, induce the dissociation of biomolecular assemblies, definitely if they are stabilized by electrostatic interactions. A way to test such an effect, is to chemically crosslink the (nucelo-)protein complexes using glutaraldehyde.

- I would suggest to run control experiments in order to check where the "unidentified objects" are coming from. Buffer solutions with added magneisum can become contaminated over time, due to growth of some micro-organisms (especially if acetate salts are used). Therefore, I would recommend to make new buffers every few weeks, and store them at 4 degrees.

- In addition, I would recommend trying surface modification of mica with a polycation. This would minimize the variable batch-to-batch surface chemistry of muscovite mica (which has also been mentioned by Remco Verdoold). If In my experience, coating the mica with a low molecular weight poly-L-lysine solution prior to sample deposition, is a very reproducible approach to study protein-DNA interactions. A protocol can be found here: Nucleic Acids Res. 2003 Nov 15;31(22):e137.

I would also be happy to have a look at your images, maybe this would be helpful?

Good luck with the experiments!

Willem

Mica is basically a poly silicate which can 'break' in nice flat layers. For electron microscopy it is essential to have an extremely clean surface before modifying it. Alternatively you could try a one side polished silicon wafer (which will give nice conductive surface as well eliminating the gold sputtering step).

For cleaning the surface I can suggest the following protocol:

Sonicate your samples for 7 minutes in MQ water and repeat again with fresh MQ after the 7 minutes.

Clean slides in piranha solution (3 parts H2SO4 + 1 part H2O2, this extremely corrosive and use with care!!) for 15 minutes at temperature of 100 degrees C.

Rinse 2x with MQ water

Sonicate again with MQ water for 15 minutes

Blow dry them with N2 gas (dust free!!!)

Bake slides in oven at 120 C to remove water

Now the slides are clean

How do you want to deposit the DNA-protein complexes on the surface?

You could make a 50 nm gold layer and use thiol functionalized DNA strands to capture the Proteins (and block the remaining surface with thiol-PEG3, polypure, Oslo).

Alternatively you could just spot them and hope they stick, but washing with very low salt buffers (to prevent salt crystals in SEM). However rinsing with MQ could damage the DNA-Protein interaction, perhaps you could label your proteins with a gold nanoparticle with homogeneous size. This eases SEM imaging.

dear Amna

I agree with Remco , the surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes. Scanning probe microscopy (SPM) is usually better than SEM since you do not need to have a conductive sample ( gold evaporation gives you some roughness ) .

DNA is the most charged polymer of nature. Mica has both positive and negative charge due to its complex nature. The pH of your buffer, the ionic strength, the use of divalent ions ( Calcium ) will then be crucial to have close to a zero charge of your surface. I expect you want your proteins to be adsorbed on DNA and not on the flat mica surface.

You can read this article for more details :

Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy. Methods Mol Biol. 2013;931:295-312. doi: 10.1007/978-1-62703-056-4_14.

Greetings

Eric

Yes! Dear Amna,

These mica surfaces are known for their catalytic activity for biopolymers, not only for biomolecules synthesis but for the destruction of biopolymers also, and are of prime importance for recently developed new branch, known as 'Astrobiology' . You can find some research papers where biopolymers were hydrolyzed in presence of these metal silicates.

Thanks

Thank you all!

Remco, I agree that the important step is surface preparation.

In fact I use Atomic force microscope, for have clean surface just by cleave the mica.

Eric, you are right. maybe the pH usually I use buffer with pH: 6.8 to 7.5 and in this pH the mica is negative charge, divalent ion (Mg++).

Unfortunately using SEM for DNA-protein complex you can get image but with low resolution, other way around is to get high resolution but you will be able to see the DNA or the protein complex.

Regards,

Amna

Dear Amna,

I know Mica is commonly used in AFM because the low surface roughness. I used it but disliked the unstable quality and moved to one side polished silicon wafers. As for the SEM you could try to find a collaborator with a High-res SEM, Helium Ion Microscope or STM.

Using AFM I got very nice images with high resolution and by applying statistical analysis I had a good results. my problem is that in some of my images I have small molecules which I am trying to understand if this molecules came out through preparing the samples or they are due to salt-surface interaction or protein complex-surface interaction.

Yes using other techniques will be great and can be the second step after I understand my results of AFM images.

Thanks Remco.

Dear Amna,

I am using AFM for the study of protein-DNA interactions as well. Some thoughts concerning your questions are summarized below.

- Freshly cleaved mica is negatively charged when exposed to water, and in buffers with high ionic strength, an extensive accumulation of cations near the interface will take place. This in turn can, in principle, induce the dissociation of biomolecular assemblies, definitely if they are stabilized by electrostatic interactions. A way to test such an effect, is to chemically crosslink the (nucelo-)protein complexes using glutaraldehyde.

- I would suggest to run control experiments in order to check where the "unidentified objects" are coming from. Buffer solutions with added magneisum can become contaminated over time, due to growth of some micro-organisms (especially if acetate salts are used). Therefore, I would recommend to make new buffers every few weeks, and store them at 4 degrees.

- In addition, I would recommend trying surface modification of mica with a polycation. This would minimize the variable batch-to-batch surface chemistry of muscovite mica (which has also been mentioned by Remco Verdoold). If In my experience, coating the mica with a low molecular weight poly-L-lysine solution prior to sample deposition, is a very reproducible approach to study protein-DNA interactions. A protocol can be found here: Nucleic Acids Res. 2003 Nov 15;31(22):e137.

I would also be happy to have a look at your images, maybe this would be helpful?

Good luck with the experiments!

Willem

Thanks Willem, in fact I tried to use mica treated with Poly-L-ornithine in my previous study using AFM imaging in air and by doing statistical analysis I found out that mica treated with Poly-L-ornithine effect the DNA molecules confirmations and the interation of DNA-protein complex.

- for keeping my protein complex stable I use Glycerol.

-I agree that using old buffer can have micro-organisms so I usualy use fresh buffer and I filter small a mount to use immediately as well I don't use 'acetate salts'.

I have attached one image for 500bp dsDNA that interact with protein complex 'the spots on the DNA molecules are represting the protein complex, the green spots on the surface with size (2-6 Angstrom) that annoy me'!

Dear Amna,

- About the “green spots”:

It might well be that the “green spots” on your surface represent unbound protein. It should be relatively easy to confirm this by making a sample with the protein alone (without DNA), and doing a volume analysis of the particles. If the volume distribution of the particles detected in this sample corresponds to the volume distribution of the features you see in the sample with DNA, chances are high that they are indeed unbound protein. Alternatively, the “green spots” are residual salt deposits, which are not removed completely during sample rinsing. In this case, a comparison with control samples (DNA alone, buffer alone) should answer your question. If the DNA solution appears contaminated, you can easily purify it (I normally use a "PCR cleanup kit").

- About DNA adsorption on freshly cleaved versus modified mica surface:

On adsorption from 3D to 2D, dimensionality is reduced, and this has a great effect on the conformational properties of DNA chains. In the framework of the worm-like chain model for DNA elasticity, the persistence length (let’s call this P) is defined to quantify the chain stiffness. Importantly, the 3D-2D transition apparently doubles P. However, during the process of adsorption, and because diffusion in 3D is orders of magnitude larger as compared to surface (2D) diffusion, an intermediate – the initially adsorbed state – conformation is formed which resembles a 3D-2D projection. This intermediate is mechanically stressed because of the apparent doubling of P, and aims to relax/ equilibrate on the surface. The speed of this process is governed (mainly) by DNA-mica friction. If the AFM sample preparation step (drop casting – rinsing – drying) is much faster as compared to the time required for equilibration, the DNA chain becomes “kinetically trapped”. If you would be interested in the DNA bend angle induced by a protein, conditions of kinetic trapping will affect the mean and the variance of the bend angle distribution, rendering the data useless. Instead, conditions of equilibrium adsorption do allow for a correct determination of the bend angle (mean and variance).

For a long time, it was thought that deposition of DNA/nucleoprotein complexes on bare mica in the presence of Mg++ results in equilibrium adsorption, whereas deposition onto positively charged, modified mica results in kinetic trapping. However, the groups of Finzi and Dunlap have demonstrated that even in the latter scenario, equilibration does occur, at least on short length scales (Biophys J. 2005 Oct;89(4):2558-63). The take-home message of this paper, is that the electrostatic contributions of the DNA structure are reduced on positively charged surfaces by partially neutralizing the phosphate backbone. This in turn renders the DNA more flexible (lower P) and makes it look more “compact” in AFM images. Nevertheless, they could show by statistical chain analysis that the DNA is still able to equilibrate! Thus, long story short: even on modified mica (poly lysine, poly ornithine,…), DNA can equilibrate and protein-induced bend angles can be quantitatively addressed.

Sorry for the long text – hope it makes some sense.

Best,

Willem