Biological sample lysis and extraction using the Covaris LE220+ for LC-MS

You will need to dilute the S-Trap lysis buffer to a working stock concentration.

Note

S-trap lysis buffer (10% SDS) is provided in the orange trays on bench.

Using the 1mL pipette, add 500µL of 2x stock of S-Trap lysis buffer to 500µL of LCMS grade water.

This is the working stock concentration (5% SDS, 50millimolar (mM) TEAB 7.5).

Add S-Trap lysis buffer (5% SDS with 50millimolar (mM) TEAB 7.5).

If using the 520045 tubes, add approximately 50µL to 130µL of buffer, the final volume will depend on the amount of sample already present in the tube.

If using the 520185 tubes, add up to 500µL of buffer , again this is dependent on the amount of sample that you wish to process.

Add the Covaris tubes to the respective stainless steel rack.

Note

According to Covaris, all spaces in the row must be filled, there are spare tubes beside the machine for this purpose. Tubes are processed in rows on the width of the plate.

Clamp the lid on the rack finger-tight, and keep the samples in the rack On ice until you are ready for processing.

On initial training, we will provide you with a general method that should be sufficient to lyse your sample. At the end of the process, if you feel that your sample would benefit from more treatment, simply run the program again.

Note

Log onto the LE220+ focussed ultrasonicator and check that the “run” box in the top right hand corner is green. Sonolab software controls the LE220+ system.

If not, it may be that the system is not at temperature. Click the “System status” box on the right hand side.

– if a red “X” is next to temperature, then you must wait until the system reaches the temperature that you have set the system to in your method before you begin. The default temperature for the system is 10°C.

How will you know if your sample is ready?

With cell suspensions, the sample will turn from opaque to transparent, a sign that the cells have lysed.

With tissue, after a quick centrifuge spin, you will see that your pellet is less than before.

Sonolab will automatically select the last method run by the user.

To select an alternative method, use the drop-down menu to load an alternative method, it is located on the HOME tab, in the top left corner.

To specify the rows of the plate that you wish to process, click “edit”.

This will open up the edit parameters page.

If you have two rows of tubes to process, click on the process in the left hand side.

Click on the two rows that you wish to process.

Click save and return to the main menu.

The Run method will remain grey and inactive until a method is selected and all necessary conditions are met.

On the right hand side, in the “Home” tab, take a look at the “System status” tab – if it is shaded red and contains a red X, then a starting parameter(s) has not been achieved – click on the button to see the issue.

If your method is set to 6°C, simply wait until the temperature has been reached.

Click on the “Motion Control” box, and click on “load”, the plate holder will move to the front of the LE220+ for plate loading.

Open the door of the LE220+ by simultaneously pressing the green door lock button while gently pulling the handle of the safety enclosure Perspex door.

Load the plate with the orientation that you specified in the method.

Note

When the “Run” button is green, the selected method may be started. Click the “Run” button.

When the method has finished, the message “Method Complete” is displayed. Click “OK”, then press the green door lock button and open the safety enclosure door. Remove the treated sample plate and place On ice.

Label 0.5mL or 1.5mLtubes for each 520045 or 520185 tube that you are processing, and transfer the lysed sample extract to the labelled tube.

If finished, logout of the Sonolab software and return the rack to the side of the LE220+ for the next user.