TITLE: Biological sample lysis and extraction using the Covaris LE220+ for LC-MS
AUTHORS: ronan.ocualain

[DESCRIPTION]
This protocol details the procedure of sample lysis and protein extraction using the Covaris LE220+ ultrasonicator.

[BEFORE_START]

 Initial preparation:

Before you begin:
Log onto the LE220+ and check that the “run” box in the top right hand corner is green. 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.



[GUIDELINES]
Initial assumptions and preparation:

Allow approximately 30 min for sample lysis and protein extraction.
You may pre-book the use of the LE220+ machine beforehand on PPMS.
If it is your first time using the LE220+, let us know at bioms@manchester.ac.uk, you will be provided with training, in which you will be provided with login rights to the machine and some basic processing methods to get you started. 
You have cells or tissue in Covaris tubes, on ice.

[STEPS]
SECTION: Preparing samples and LE220+
1.  You will need to dilute the S-Trap lysis buffer to a working stock concentration. 
 


Using the 1mL pipette, add 500 µL of 2x stock of S-Trap lysis buffer to 500 µL of LCMS grade water.
This is the working stock concentration (5% SDS, 50 millimolar (mM) TEAB pH 7.5).
 

SECTION: Preparing samples and LE220+
2. Add S-Trap lysis buffer (5% SDS with 50 millimolar (mM) TEAB pH 7.5).
 

SECTION: Preparing samples and LE220+
2.1. If using the 520045 tubes, add approximately 50 µL to 130 µL of buffer, the final volume will depend on the amount of sample already present in the tube.

 

SECTION: Preparing samples and LE220+
3. Add the Covaris tubes to the respective stainless steel rack.
 

SECTION: Preparing samples and LE220+
4. Clamp the lid on the rack finger-tight, and keep the samples in the rack on ice until you are ready for processing.





SECTION: Preparing samples and LE220+
2.2. If using the 520185 tubes, add up to 500 µL of buffer, again this is dependent on the amount of sample that you wish to process.
SECTION: Sonolab 8.2 software
5. 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.


 

SECTION: Sonolab 8.2 software
6. Sonolab will automatically select the last method run by the user.
SECTION: Sonolab 8.2 software
5.1. 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.
SECTION: Sonolab 8.2 software
5.2. With tissue, after a quick centrifuge spin, you will see that your pellet is less than before.
SECTION: Sonolab 8.2 software
7. 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.
SECTION: Sonolab 8.2 software
8. To specify the rows of the plate that you wish to process, click “edit”.
SECTION: Sonolab 8.2 software
9. This will open up the edit parameters page.
SECTION: Sonolab 8.2 software
9.1. If you have two rows of tubes to process, click on the process in the left hand side.
 

SECTION: Sonolab 8.2 software
9.2. Click on the two rows that you wish to process.
 

SECTION: Sonolab 8.2 software
9.3. Click save and return to the main menu.
SECTION: Sonolab 8.2 software
10. The Run method will remain grey and inactive until a method is selected and all necessary conditions are met.
SECTION: Sonolab 8.2 software
10.1. 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.
 

SECTION: Sonolab 8.2 software
10.2. If your method is set to 6 °C, simply wait until the temperature has been reached.
  

SECTION: Running your method
11. Click on the “Motion Control” box, and click on “load”, the plate holder will move to the front of the LE220+ for plate loading.
SECTION: Running your method
12. 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.
SECTION: Running your method
13. Load the plate with the orientation that you specified in the method.

 

SECTION: Running your method
14. When the “Run” button is green, the selected method may be started. Click the “Run” button.
 

SECTION: Running your method
15. 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.
SECTION: Running your method
16. Label 0.5 mL or 1.5 mLtubes for each 520045 or 520185 tube that you are processing, and transfer the lysed sample extract to the labelled tube.
 



SECTION: Running your method
17. If finished, logout of the Sonolab software and return the rack to the side of the LE220+ for the next user.
 
  

SECTION: Preparing samples and LE220+
2.