Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

The overall goal of this procedure is to image small and asymmetric protein using a high throughput optimized protocol of negative staining electron microscopy. This is accomplished by first incubating the protein in a prepared incubation station while preparing a negative staining workstation. The second step of the procedure is to quickly wash the sample on three water droplets sequentially.

The third step is to carefully stain the sample on three stain droplets sequentially. The final step is to remove the excess solution by blotting from the backside of the EM grid and then gently drying under nitrogen gas. Ultimately, results can show high resolution images of small proteins through TEM imaging under a low defocus condition.

The main advantage of this technique over cryo electro microscopic is set. It can allow for high throughput examination and high contrast imaging of small and asymmetric protein structures while reducing the structure artifacts from conventional negative staining. Though this method can provide insight into the structural basis for small protein mechanisms like the cholesterol ester transfer protein.

It can also be applied to other proteins such as IgG one antibody high density lipoprotein and proteasome, which vary widely in size. Visual demonstration of this method is critical as the washing and staining steps are difficult to learn because of variation in timing and even sample blotting can cause dramatic effects in the finished stained protein Demonstrating the procedure will be missed among Z.A research fellow from my laboratory. When preparing for this protocol, it is necessary to make 1%urinal form eight solution with utmost attention paid to minimizing its exposure to light.

This includes wrapping the syringe N 0.2 micron filter parts in foil when it is time to first filter the solution. After filtering, store two milliliter foil wrapped vial aliquots at negative 80 degrees Celsius on the day of the experiment. Thaw an aliquot in a room temperature water bath once completely thawed.

Filter it again through a 0.02 micron filter with parts wrapped in foil. The collection vial should also be wrapped in foil. Transfer the negative stain to an icebox until it is used.

Make a droplet holding sheet by pressing an eight inch length of param onto a pipette tip supporter. This makes road indentations that serve as five millimeter diameter wells. After making six rows of wells, flatten the ice on the surface of a styrofoam tray, then position the sheet onto the ice.

Next, add 35 microliters of DI water into the first three wells of the sheet on the left side. Then load the three right wells in the sheet with 35 microliters of the prepared negative stain solution. Cover the tray to minimize light exposure to the solution.

This will serve as a staining station. Now prepare an EM grid incubation station from an empty pipette tip box with an attachment onto which tweezers can be mounted. Fill the box halfway with ice so that the ice surface is close to the tip of the tweezers when they're mounted.

First glow discharge the thin carbon coated 300 mesh copper EM grids for about 10 seconds. Next, pick up a grid with tweezers that hook onto the EM grid incubation station. Hang the tweezers with the grid, so the grid is at a 45 degree tilt a half inch above the ice.

Now dilute the protein sample in DPBS at 50 to 100 micrograms of protein per milliliter of solution. Immediately apply about four microliters of the dilution to the EM grid. Allow the sample to incubate for about a minute.

After one minute, quickly dab the mesh with filter paper to remove excess solution. And then at the staining station, touch the mesh to the first drop of water on the para film. Quickly repeat the process of drying the filter and retting it with the next droplet.

Use a total of three water droplets and do this as quickly as possible. Washing the grid with water must be done rapidly to avoid adverse effect of buffer change to the protein. After dabbing off the last wash within three seconds, start floating the grid on the first drop of negative staining solution.

Let it float there for 10 seconds. Meanwhile, clean the tweezers by pressing the tips into filter paper. A few times after the ten second float blot off the solution with filter paper and start another float in the next droplet of stain for two seconds.

Clean the tweezers during the two seconds. Next, blot the grid dry and put it onto the third droplet. For a full minute cover the staining station during this step.

Removing the stain must be done by touching the backside of the EM grid for a precise amount of time to ensure the grid is dried evenly. If the filter paper touches the stain for too long, then too much stain could be removed leading to poor imaging. Next, proceed with drying the grid.

First, touch the entire non-carbon side to the filter paper until the solution transfers off. Second, apply a gentle stream of nitrogen gas. Now transfer the grid to a dish lined with filter paper and partially cover the dish.

Allow the grid to dry in the dish for 30 minutes at room temperature. Alternatively, lipid containing samples can be baked at 40 degrees Celsius for an hour. Once fully dried, transfer the EM grid to an EM grid storage box before beginning first, align the TEM.

Check the resolution of the highest visible thaw ring with the power spectrum of a carbon film area of the negatively stained grid, which should be better than the targeted resolution. Then to adjust the alignment carefully check the power spectrum on the carbon film area of the specimen. Under a properly aligned condition, more than 20 T tho rings can be visualized, which corresponds to visualization better than five angstroms.

The rings were made by furrier transfer of an amorphous carbon image under a defocus of 1.6 microns and a dose of 20.4 electrons per square.Angstrom. Now bring the focus back to a near Scherzer focus condition about 0.1 micrometers for small proteins imaging during examination of the EM grid. Ideally, stained areas are usually near the edge of thicker stained cloudy areas at low magnification.

The structure of lipoprotein specimens were viewed with OPNS. Examples include recombinant HDL, human plasma, LDL, human plasma IDL, and human plasma VLDL. Smaller structures like 53 kilodalton, CETP were also viewed.

This is one of the smallest proteins successfully imaged by eem. Super imposition of the CETP crystal structure matches the image very well, very dynamic. Heterogeneous proteins were also viewed in 160 kilodalton immunoglobulin G antibodies.

Three subdomains were clearly visible. Closer examination of the IgG one antibody was used to make a comparison to its crystal structure. The crystal structure of the IgG one antibody matches with the EM view of these antibodies in many ways, such as in the domain positions and their shapes.

Other viewed molecules include 28 kilodalton, lipid free apo lipoprotein A one grow EL, and proteasomes. Essentially, the OPNS method significantly advances the boundary of EM imaging for small asymmetric structures as well as associated mechanistic studies While attempting the procedure. It's important to reduce light exposure to the stain reagent as much as possible to quickly wash the specimen and use near treasure focus for imaging Following this procedure.

Other methods like electron tomography can be performed in order to answer additional questions like the nanometer resolution structures of single molecules and the confirmational changes among them. After this development, this technical paved the way for researchers in the field of the structural biology to explore the magnets of a cluster ester transfer among the human plasma lipoproteins.