Application 1: Protein intrinsic Fluorescence for Crystal Detection or distinguishing Protein Crystals from Salt Crystals
Example 1: Protein crystals among ammonium sulfate crystals
Example 2: Pure salt crystals:
Example 3: Crystal screening results:
Application 2: RNA and Aptamer Identidication applying the Nucleic Acid specific Dye "SYBR-GOLD":
Example 1: Distinguishing RNA from Salt:
Example 2: Aptamer-RNA Complex Identification:
Trace Labeling Procedure
Example: Sample in green light
In Situ DLS Applications
- Angle Depending
- Temperature Depending
- Classic DLS
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Highly oligomerized sample
Partially aggregated sample
A)Protein Purification: After purification, a sample might not be in the desired aggregation state or a biological relevant complex of molecules has not formed yet. Some buffer conditions might support complex formation apart from its natural environment but often such conditions are unknown.
B)Condition Screening: One strategy to obtain biological complexes is the application of various conditions (e.g. sparse matrices) systematically to a sample by adding corresponding buffers. However, a manual pipetting procedure is a tedious task and errors are likely. In order to even the workflow, automated dispensing systems (like the Oryx8) are available enabling a time, material and manpower efficient way to apply such buffer matrices. Usually, standard microbatch plates and samples sealed under inert paraffin oil are used.
C)Sample Qualification: However such an approach results in many conditions (usually 96) exceeding grid holding capacities of most grid carriers by far. Furthermore, most of the applied conditions have a negative effect on the sample, leading to a very inefficient usage of a cryo-EM when all samples would be transferred to grids and investigated by cryo-EM. A key technology to select samples for subsequent cryo-EM analysis is in situ DLS (performed with the SpectroLight 600). It allows to identify suitable buffer conditions by particle size determination, non-invasively and directly in wells of a microbatch plate. Size and dispersity determination enables identification of the few conditions that stabilizes the desired macromolecular complex.
D)Sample Transfer: The few good conditions can be selected for subsequently investigation for cryo-EM, resulting in a significant increase of positive hits on the grids.