Honest Protocols! Making Lysates

 This is the second article in my new series; Honest Protocols, where I give a protocol for common lab techniques along with the realities of the protocol and the emotional changes they cause.

This week, I'm looking at how a lysate is made. 

A lysate is a liquid mixture that contains cell proteins, DNA and some other molecules usually contained within the cell- but it doesn't have the living cells itself.  This is done by breaking the cells open - called lysing and letting the molecules out. For most people, a lysate is often used for a western blot, but you can also use it for DNA extraction and protein purification. Lysates can be frozen so in theory you can forward-think and freeze some lysates in advance. Still, you have to make them at some point:

1. Pretend you are doing a simple splitting, and you aren't preparing for a western blot -one of the most annoying techniques that was ever invented. Decide to actually forward think for once - book a slot on the cell hood and remember to take your media, trypsin and PBS out of the fridge. 

2.Locate your cells in the incubator.  Look at them via a microscope, confirm there is enough of them and start preparing to split.

3.Carry out steps 10-16 from honest protocol 1 - Splitting cells. 

4. Find that this time, it’s not the flasks that have run out, it's the 15 ml tubes. 

5. Swear, run across to the storeroom, grab a bag of 15 ml tubes and run back to the cell culture hood.

6. You also find that the Eppendorf tubes* have run out and it never occurred to you to check them before you went to the storeroom. 

*Eppendorf tubes are tiny plastic tubes that are designed to have secure locks and seals to stop any contamination. You can get multiple types but the most common hold about 2mls of liquid. You can freeze them and centrifuge them. 'Eppendorf tubes' are a mouthful so you can just call them E-tubes. 

E-tubes. They also come in a load of colours. 

7.Stomp back to the storeroom, grab a bag of E-tubes and stomp back,

8.aYou can't work out how to open the bags neatly and efficiency, so just break it open with your thumb, grab what you need and spray it down with ethanol.

9. Repeat step 16-20.  Resuspend pellet with 1ml of PBS and transfer to an E-tube.

10.Prepare RIPA- protease inhibitors* mixture in a 100:1 ratio. Try to remember how to calculate ratios. Remember that you are going to need to chill the centrifuge* in advance so go and run it at 4 degrees for 10 minutes.

*RIPA buffer is what we use to get the protein out of the cell membrane and any tissues. It's a lysis buffer so it dissolves the cell membranes and releases the protein. But this process also causes natural enzymes called proteases to be released and these destroy the proteins. So, we add protease inhibitors to stop the proteases from acting. 

*The centrifuge being used here is different from the smaller one used in cell culture. Here, you are using a benchtop centrifuge which is much bigger. This gives it a much larger rpm, and you can also adjust the temperature. For this, you want the temperature to be low to stop the proteins from getting deformed. So we call it a refrigerated benchtop centrifuge. 

A refrigerated benchtop centrifuge. 

11. Look at the size of the pellet and attempt to judge how much of the RIPA-Protease you should add. You're supposed to add 'just enough' to resuspend the pellet. Eventually decide on 100 micro litres and call it a day. 

12. Vortex the E-tubes. Stick your finger in it when no one is looking.

A vortex. It's basically a mixer and

 makes sure the pellet is totally resuspended. 

Everyone sticks their (gloved) finger in it 

at some point.

13. Try to remember what rpm you're supposed to centrifuge at. Settle for 12,500 rpm for 10 minutes.  Brace yourself for the horror that is an unbalanced centrifuge.  Internally scream ‘KEEP THE LIQUID!' at yourself. 

14. Pipette liquids into fresh E-tubes. Realise that you really don't want to start your western blot today, so you decide to just freeze them for tomorrow. 

15.Discover that your cryobox* is full.

*Cryobox is just a fancy word for plastic boxes that store e-tubes and can resist high temperatures. 

                                            A cryobox. 

16. Work out what you can throw away in the ice storage box.

17. Stuff the e-tubes into the new spaces.

18.With the help of a coffee, return to your desk and start mentally preparing yourself for the fun that is a western blot.