Negative-stain TEM is an invaluable tool for assessing the purity, homogeneity and morphology of large (> ~100kDa) macromolecular assemblies. It is also a routine tool for imaging tissue (eg histology & pathology). The major research questions that we often address using negative stain TEM are:
Sample assessment, morphology and ultrastructure.
The most commonly asked research questions that we get in the EM unit are to do with basic characterisation of biological complexes. Is a sample pure and/or homogeneous? What is a samples morphology? For example, is a sample of a potentially amyloidogenic protein fibrillar or does it contain amorphous aggregates? Is a preparation of a virus monodisperse or aggregated? Are all the virus particles intact? Such questions can be rapidly and unambiguously answered with simple, negative-staining EM imaging. For negative-stain TEM, the sample is adsorbed to an EM grid bearing a continuous carbon film (we can make or supply this). The sample is then embedded in a layer of a heavy metal salt (routinely we use uranyl acetate, but we can also supply tungsten and molybenum stains). This stained layer is then dried, and imaged. Negative stain EM generates high contrast in the microscope and can provide a clear, quick, visual answer to these questions.
Negatively-stained amyloid fibrils of β-2-microglobulin fibrils.
Domain mapping experiments on the motor protein dynein. Insertion of a GFP domain at known points in the sequence of dynein, allows the architecture of the dynein head to be mapped. Courtesy of Dr. S.A. Burgess.
Structure and conformational change by negative stain EM
Negative staining generates high contrast that is essential for imaging relatively small molecules (>100KDa). The EM unit in Leeds has a vast experience in imaging small, dynamic proteins such as the molecular motor proteins myosin and dynein using negative stain-EM. Such experiments can provide invaluable insight into protein structure.