How to build miniaturized purification workflows in Synthace
Learn how to get started with building a miniaturized purification workflow in Synthace.
Note: This workflow exists in your Synthace environment. Click on Tutorials > Getting Started with Miniaturized Purification: RoboColumns > Protein Purification Workflow with Collected Fractions to view it on your own.
This is an example of a generic miniaturized purification workflow. Your own process may differ in terms of the number of chromatography action steps, reagents used, etc.
Note: This article focuses on workflow building. To learn more about the RoboColumn elements, click here. To learn more about the core elements, click here.
In miniaturized purification workflows, there are several types of connections used to wire elements together that facilitate the flow of information:
Robocolumn connections ensure each RoboColumn element “knows” where the previous element left off, in other words, what is the state of the RoboColumns
Buffer connections ensure each RoboColumn element “knows” which buffers/samples are available for loading
Plate connections allow you to pass over information regarding the collection/gradient plate names and plate types as well as which wells have been used already. In this manner you don’t need to manually input the information in each Run Chromatography stage element.
Define RoboColumns and reagents
In the first instance, you need to tell Synthace about your starting materials such as the RoboColumns, buffers and samples in your purification protocol, before you can start adding chromatography stages that will be using them.
All workflows will start with using Define Resin Types, Define RoboColumns Plate, and a Define Liquids and Plates elements.
The output of Define Resin Types is connected to the input of Define RoboColumns Plate so the latter knows which resins you have available for allocating to the RoboColumn array plate.
The Define Liquids and Plates here describes ALL buffers that will be used during your purification workflow. Use this element to also fill in relevant metadata such as subcomponents of buffers and their concentrations.
Note: You may also define the samples you are purifying here as well, however in this workflow the samples are defined using a separate Define Liquids and Plates element as described in the Load Samples section.
Equilibrate
After defining your RoboColumns and reagents you plan on using, connect the outputs of the Define RoboColumns plate and Define liquids and plates into the inputs of the first Run Chromatography Stage element to provide the liquids that are available for loading.
Fill in the Stage name and click on Load Parameters to configure or view the chromatography actions. To learn more about the Load Parameters elements, click here.
Most miniaturized purification workflows will start with either a cleaning or equilibration step. Typically you would not be interested in collecting the flow through from this step. By leaving Collect Flow Through unchecked**,** the loaded liquids will go to waste.
Load Samples
After cleaning or equilibrating the RoboColumns, you can load your sample into the RoboColumns. You can achieve this by adding another Run Stage Chromatography element, filling in the stage name and altering the inputs slightly to indicate to Synthace that you are loading a sample.
Connect the RoboColumns After output of the preceding Run Chromatography Stage element to the RoboColumns Before input of the new Run Chromatography Stage element to pass along information about the state of the RoboColumns.
The defined sample is fed into the Available Buffers/Samples to load input. This can either come from a new Define Liquids and Plates element (as it is in this example) or from the Remaining Buffers/Samples to Load output on the preceding Run Chromatography Stage element. It depends on how you defined your samples.
IMPORTANT: When defining your sample, you must add the metadata "Role = Sample". This is important for meta-data tracking and data processing.
Tip: If you wish, you can also define the Molecular Weight (in Da) and Molar Extinction Coefficient (in M^-1cm^-1 ) **** of your protein of interest. This will then be used to automatically calculate downstream purification metrics, such as concentrations of collected fractions and yield. To learn more about automated purification analysis in Synthace, click here.
Since you are loading a sample, you may want to collect fractions. Toggle on Collect Flow Through and set the appropriate collection settings.
Wash
Now that the samples are loaded, it is time to remove unbound material by loading the wash buffer through each RoboColumn by adding yet another Run Stage Chromatography element, and giving it a unique stage name.
Since Wash buffer was defined in the first Define liquids and plates element (in the Define RoboColumns and reagents section) that we connected to the first Run Chromatography Stage (Equilibrate step), we want to wire the Remaining Buffers/Samples to Load output from this element, rather than the Load step which only knows about the samples, to the Available Buffers/Samples input of the new Run Stage Chromatography element.
Connect the RoboColumns After output from the preceding Run Chromatography Stage element in the Load Samples section to the input of the new Run Chromatography Stage element.
Since we also want to collect fractions in this stage, continuing where we left off in the Load stage, connect the Collection Plate Name Used, Collection Plate Type Used, and Collection Wells Used outputs from the preceding Run Chromatography Stage element in the Load Samples section to the appropriate inputs of the new Run Chromatography Stage element.
As a result some collection settings are now pre-populated. Synthace will use the remaining wells available on the first plate for fraction collection in this stage.
Tip: If you want to collect fractions in a new plate, do not pass the Collection Plate Name Used, Collection Plate Type Used, and Collection Wells Used outputs between elements and instead define a new plate in the Collection Settings.
Elution
To elute your molecules of interest from the RoboColumns, add another Run Stage Chromatography element and give it a unique stage name. Once again connect the appropriate RoboColumn, liquids to load, and plate collection information outputs from the preceding stage (in this case Wash stage) to the new Run Chromatography Stage element. Set up your loading parameters and collection settings (if not pre-populating through a connection).
Strip
Keep adding Run Stage Chromatography elements for every additional stage you need. After eluting, load Strip buffer to the RoboColumns to remove any excess material left over from elution and collect fractions, ensuring appropriate connections are in place to the element from the preceding chromatography stage.
Re-Equilibration
Add another round of Equilibrate buffer to your RoboColumns. Note this time that there is no need to connect the collection plate outputs from the previous Run Chromatography Stage element since you do not plan on collecting fractions.
Note: Each purification stage name needs to be unique. Equilibrate 2 is the Purification Stage Name used here since this is the second equilibrate stage in this workflow.
Storage
For the final stage of this workflow, add a storage buffer to the RoboColumns, without collecting fractions.
