The overall goal of this procedure is to visualize purified protein samples by negative stain electron microscopy. This is accomplished by first preparing the electron microscopy grids that will support the sample. The second step of the procedure is to prepare the negative stain solution.
Next, the sample is applied onto the electron microscopy grid and negative stained. Lastly, the EM grid is loaded into an electron microscope, and images are recorded under appropriate optical conditions. Conventional brightfield, DEM images are high contrast and sharply detailed, showing single macromolecule scattered over a featureless background.
Negative stain electron microscopy is a simple method of visualizing your favorite protein. It can be used as a routine assay to examine your purified protein, either for structural analysis or other biochemical and biophysical assays. Here we demonstrate how to prepare a negatively stained sample for electron microscopy.
Visual demonstration of this method is helpful as some of the steps are difficult to learn from text descriptions and figures. Start by using a glass pipette to place a single drop of collodion into a large beaker of water on the water surface. As the collodion spreads, it may capture dust from the water surface.
Remove and replace a dusty film of collodion plastic with another drop. Next place between 50 and 100 EM grids one by one onto the floating plastic film shiny side down. Select 400 mesh grids for routine negative stain and select 200 mesh grids.
For collecting tilt pair images. Select a piece of slow absorbency unwrinkled paper that is slightly larger than the area of the grids. Place the paper on top of the grids and wait until the paper becomes completely wet.
The paper should not absorb water too quickly. We use paper from a cheap notepad. The grids are now sandwiched between the plastic film and the paper.
Use a pair of forceps to place the paper in a Petri dish to slowly air dry. Avoid faster drying methods as they can lead to wrinkles. The carbon coating machine used in our laboratory is a Creston 2 0 8 C high vacuum turbo carbon coer.
Equipped with a Creston film thickness monitor. The monitor utilizes a quartz balance that is sensitive to carbon deposits. However, the protocol described here does not require this device Begin by sharpening and loading the carbon rods in a carbon coating machine.
Next coat, a glass slide with vacuum grease leaving half of the frosted area uncoated After carbon coating, the greased regions of the slide are used to estimate the thickness of the carbon coat. Place the paper with grids on the stage under the bell jar of the carbon coating machine. Position the greased glass.
Slide next to the grids. Pump the vacuum chamber up to at least 10 micro tours otherwise sparking will generate many large particles in the grid. Now increase the current very slowly.
The sharp tip of the carbon rod will become extremely bright. Do not observe it with the naked eye as the coating begins, the glass slide will change color to a dark gray as the carbon coat builds up, cut the current at the desired carbon thickness and take into account the transparency of the bell jar. When making this judgment, Once the darkness of the slide is calibrated to the carbon thickness, this method is as accurate as the more costly film thickness monitor.
When the process is finished, vent the vacuum chamber and remove coated grids for later use. The thin plastic layer will not affect the quality of the negatively stained image, but can be removed by soaking the grids in chloroform for several hours Before proceeding. Make fresh urin L formate solution or use solution that has been stored in the dark for less than two weeks.
Good fresh solution has a yellow color, a pH of around four and runs very clean with no precipitates. Begin by using a commercially available system to glow. Discharge the carbon coated grids.
Next place, two 20 microliter drops of distilled water and 2 25 microliter drops of fresh urinal formate solution onto a piece of perfil. Using reverse force anti capillary forceps, hold a glow discharged grid, carbon coated side up. Critically, these forceps do not siphon liquid from the grids.
Now apply two microliters of protein sample to the grid and wait while the proteins are absorbed into the grid. This typically takes 30 seconds, but very dilute samples will require more time, which can impact buffer concentrations. When finished, blot off the protein sample using filter paper.
Then wash the grid twice by briefly touching its surface to a drop of water and blotting it dry within a few seconds. After the water briefly touched the grid to a drop of stain and quickly blot it dry. Using the same technique, allow the grid to be in contact with a second drop of stain for 20 seconds.
Finally, air dry the grid on the bench fume hood, or preferably in a vacuum before the EM session, check the alignment of the EM microscope loading a perfected alignment file if available, should restore it to near perfect condition. Now load the EM grid on a single tilt standard specimen holder. Next, insert the holder into the stage.
Then wait for the pumping cycle to complete and insert the holder into the column. Now begin searching for a sample area with good stain. Manually focus using the minimum contrast method or by using a CC, D camera and computing the live four.
Your power spectrum. Then set the deep focus as needed, usually to negative 1.5 microns. Stain concentration can vary across the grid, so search for proteins with white contrast.
Avoid areas with uneven stain or positive stain where protein contrast becomes black. Using the outlined approach to making grids and visualizing negatively stained samples. Several familiar proteins were imaged for demonstration purposes.
Take note of their detail and sharp contrast. After watching this video, you should have a good understanding of how to generate samples of negatively stained macromolecules for observation in an electron microscope. Once mastered, this technique can be performed in about four hours.
Following this procedure, other methods like 3D reconstruction can be performed in order to better understand molecular structures.
