[3dem] blotting changes structure?

[Henning Stahlberg]

Dear Sheemei,

I don't think the blotting itself does any harm to the proteins, the blotting paper stays in large distances form the grid for most grid areas.

However, harm to proteins will come from drying, changing the salt concentration, or from acidification.

Marc Adrian in the lab of Jacques Dubochet once told me that some filter papers were very acidic. He used to wash the filter papers in buffer solution, then rinse, and then iron to flatten them, before using them for blotting. However, he stopped doing that in later years, as he didn't believe it made a big difference for most proteins.

Marc Adrian used self-made holey carbon grids that he coated with a thin layer of Pt-C. He did not glow-discharge the grids, but washed these grids in ethylene acetate immediately before usage, which rendered the grids semi-hydrophilic on both sides. He adsorbed a 3 ul drop of sample solution to the metal-coated surface of the grid, and then used a manual plunger in the normal lab atmosphere in a modified chemical hood with reduced air flow, to blot these grids.
He blotted the grid with Whatman No.1 filter paper from only the one side onto which he had added the sample. This resulted in the sample concentration increasing in the holes, as he did not remove any sample liquid from the back side of the grid.  He once told me that he believed that the sample particles that are adsorbed to the rear water surface in the holes of the grid were as tightly bound to that water surface as if they were bound to a sticky carbon film. By blotting only from the front, he kept all sample particles that had found their way into the holes to the rear surface, which increased the sample concentration there. However, this way he imaged particles that were exposed to that surface for some time, which may or may not have damaged those particles. He blotted by hand for roughly 3.5 seconds, and then released the grid into the ethane by gravity plunging without removing the filter paper from the grid, so that the grid was sliding down from the filter paper while dropping.

Particles may be bound to that rear water surface by charges, which is probably ok but tends to orient the particles. Or, the particles may be bound by hydrophobic forces, meaning the particles seek exposure to the air-water interface, during which they might partly denature. In the beautiful city of Lausanne in Switzerland at the lake Geneva, Marc was more successful with blotting during some seasons than others. Spring and fall were his favourite seasons, where the air humidity and temperature in the lab was most favourable for his method.

The Vitrobot instead is using a controlled humid atmosphere, blots from both sides, and then waits, before plunging, as far as I understand. This should result in imaging the bulk solution and not the surface exposed particles, so that you might end up with a lower concentration of particles. The waiting before plunging will require a well controlled humidity, which the Vitrobot provides if everything works well.

If not, i.e., if your humidity is not right, then if you blot a grid and then leave it time to dry before plunging, you will increase the salt concentration in your buffer a lot.  Or, if you work with high concentrations of salt such as when using saturated ammonium molybdate salt solution for cryo-negative staining, then any waiting between blotting and plunging will in a humid atmosphere have water condensate into the solution, so that the salt concentration in your sample is dropping between blotting and plunging.
Changes in the salt concentration would obviously be dangerous for most samples.

Greetings,

Henning.


Henning Stahlberg, PhD
Prof. for Structural Biology, C-CINA, Biozentrum, University Basel
Mattenstrasse 26 | D-BSSE | WRO-1058 | CH-4058 Basel | Switzerland
http://c-cina.org | Tel. +41-61-387 32 62



On 13 Aug 2014, at 09:18, Sheemei Lok <sheemei.lok at duke-nus.edu.sg<mailto:sheemei.lok at duke-nus.edu.sg>> wrote:

Hi all,
Does anyone have ever observed that the blotting of sample on cryoEM grid during freezing, changes the structure of your protein? Also are there other alternatives that we can employ to overcome this problem?

Regards,

Shee-Mei Lok
Assistant Professor
Program in Emerging Infectious Diseases,
Duke-NUS Graduate Medical School,
KTP Building
Level 9 Room 19
8 college Road,
Singapore 169857
Ph: 65-65165840
Fax:65-62212529
email: sheemei.lok at duke-nus.edu.sg<applewebdata://E5F18255-3202-4C1B-877E-34E4A73B3779/sheemei.lok@duke-nus.edu.sg>


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