Custom DSF experiments

These are run when you want to compare more than one protein on the same plate. Often you might be comparing the stability of different mutants of the same protein (all under the same buffer conditions) or comparing the stability of the same protein when different compounds are bound.

In these cases you will design the experiment, and dispense out the appropriate replicates of the proteins by hand (as our machines aren’t set up to work with multiple samples in the same plate). Then we add dye, seal the plate, and run the heating protocol. After the raw data have been collected, these can be analysed through the MELTDOWN program, but you will need to provide the appropriate ‘Content Map’ file.

Each well will need 20 µL of 20-50 μg/mL protein. We recommend that at least 3 replicates of each sample are dispensed.

The MELTDOWN program has two sections: a summary page (which shows the “protein as supplied” control, and a summary Tm plot, as well as information about the controls, and the overall success of the DSF run.

The remaining pages of a MELTDOWN report show the individual curves from the experiment, where similar curves are drawn on the same curve plot.

What is the Content Map file?

The Content Map is a text file which describes what is in each well of a DSF experiment, and which tells the MELTDOWN program about how to analyse the plate.

The Content Map file has a header row

Well

Condition Variable 1

Condition Variable 2

pH

d(pH)/dT

Control

Order

 

Well

Describes the well position as a (row, column) e.g. A1, B9.

Condition Variable 1

All of the DSF curves with the same Condition Variable 1 will be drawn on the same curve plot, and will be in the same column in the summary T_m graph. This Variable must not be blank.

Condition Variable 2

The curves with the same Condition Variable 1 and Condition Variable 2 will be used to calculate the statistics (Average, error estimation) for the T_m. This Variable can be left blank.

pH

This column describes the pH of the experiment, if relevant. If this is included, the Summary T_m plot will be ordered by pH. The pH column can be left blank.

d(pH/dT)

This column gives some idea of how much the pH might vary, depending on the melt temperature. This column can be left blank.

Control

This column allows the user to set a flag (’1’) to tell the program that the well is being used for a control curve. There are four controls recognised by MELTDOWN: ‘Lysozyme’, ‘No Dye’, ‘No Protein’ and ‘Protein as supplied’. A control is recognised by having one of the four types in the Condition Variable 1 column, and a ‘1’ in the Control column.

Order

This column dictates the order in which the different curves defined in Condition Variable 1 will be drawn on the Summary T_m plot.

As an example, take a case we had where we were analysing different mutants of the same protein AtzH;

We had 14 mutants to analyse, and wanted to compare their stability to the wild type protein. Each mutant was dispensed into 6 wells – so wells A1-A6 contained the same mutant, as did wells A7-A10 and so on. There were two controls included (in 6-fold replication) as well – lysozyme, and the wild type protein.

The Content Map describing the experiment can be found here

The MELTDOWN analysis can be found here