TITLE: Ribosome Purification for OnePot PURE cell-free system
AUTHORS: Konstantinos Ragios

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol we explain the procedure of ribosomes purification with the method of hydrophobic interaction chromatography (HIC) so they can be used for a PURE cell-free system.</div></div>

[STEPS]
?. [Small cell culture]
Add 100ml of LB medium to the 1L Erlenmeyer flask
Work under flame Sterilize the bench with 70% Ethanol
?. [Small cell cultu]
?. [Small cell culture]
Inoculate the LB with 10μl of A19 E. coli strain kept in glycerol stock
?. [Small cell culture]
Incubate overnight (at least 12 hours) at 37oC rotating at 250rmp
?. [Large cell culture]
Add 2l of LB medium to each of the two 5l flasks
Work under flame Sterilize the bench with 70% Ethanol
?. [Large cell culture]
Inoculate the flasks with 30ml of the A19 E. Coli culture each
To store some of the remaining bacteria: Add to the desired tubes same quantity of the bacterial sample and 40% Glycerol stock and mix.Then you may store them at -80oC for future use.
?. [Large cell culture]
Incubate the flasks for 2 hours at 37oC rotating at 180rpm
?. [Large cell culture]
Test the bacterial sample with OD600 Spectrophotometer
?. [Large cell culture]
Add 1ml of the sample to a cuvette and measure it
?. [Large cell culture]
If the measurement is lower than 1.0 continue incubating  and after the appropriate time repeat the measurement
Take into account that E.coli has a doubling time of about 20-30 minutes
?. [Cell suspension and lysis]
Centrifuge the bacterial sample for 20min with 6000 RCF at 4oC
Precool the centrifuge to 4oC  before using as the sample is quit sensitive
?. [Cell suspension and lysis]
Remove the supernatant from the tubes .The cells should be shaped into a pellet on the wall of the tube
?. [Cell suspension and lysis]
Use 40ml of the buffer to resuspend the cells and then with the another 10ml wash the tubes to collect any cells left attached to the tubes
?. [Cell suspension and lysis]
Add 25μl of β-Mercaptoethanol to 50ml of Suspension buffer
The final concentration of β-Mercaptoethanol in the buffers should always be 7mM before each use
?. [Cell suspension and lysis]
Gather the sample into two tubes (appr. 25-35ml liquid in each)
At this point we can store the samples at -80oC overnight
?. [Cell suspension and lysis]
Sonicate each tube on ice with 20s on 20s off for an active time of 4min at 70% of total power
Clean the sonicator by applying one operation cycle to milli-Q water
It is very important to keep the cells on ice to avoid damaging the proteins as they are very sensitive to temperature
?. [Cell suspension and lysis]
Centrifuge the samples for 20min with 20000RCF at 4oC
Precool centrifuge at 4oC
Debris are gathered on the walls in pellet shape
?. [Cell suspension and lysis]
Collect the sample into a single beaker and measure the total volume (e.g. 60ml)
?. [Cell suspension and lysis]
Prepare the same quantity of High salt suspension buffer by adding 2000x concentration of β-Mercaptoethanol   (e.g. add 30μl of β-Mercaptoethanol to 60ml of High salt suspension buffer) and mix the solutions.
?. [Cell suspension and lysis]
Centrifuge the samples for 20min with 20000RCF at 4oC
Precool centrifuge at 4oC
Due to the use of the High salt buffer the unwanted debris will be precipitated
?. [Cell suspension and lysis]
Filter the supernatant through a syringe filter with a 0.22μm PES membrane and gather the sample to a single Beaker
?. [Ribosome purification]
Preparation of the purifier: Clean the input filter of the AKTA Purifier                                                Set the ambient temperature to 4oC                                                Connect three HiTrap Butyl HP column (5ml)  in series to the purifierPreparation of the buffers: Add 75μl of β-Mercaptoethanol to 150ml of Buffer C                                                Add 60μl of β-Mercaptoethanol to 120ml of Buffer D
?. [Ribosome purification]
Remove the ethanol in the columns with 45ml of Milli-Q water
Set up the flow rate as 4 ml/min (Monitor the pressure while running the column and do not exceed the Max limit of pressure of columns.)
?. [Ribosome purification]
Wash the column with 45mL of wash buffer 1 (100% buffer C)
?. [Ribosome purification]
Wash the column with 75mL of wash buffer 2 (80 buffer C, 20% buffer D)
?. [Ribosome purification]
Add approximately 90% of the lysate sample to the system
?. [Ribosome purification]
Elute the ribosomes with 60ml of elution buffer 1 (50% buffer C, 50% buffer D) and then with 60ml of elution buffer 2 (100% buffer D)
?. [Ribosome purification]
The eluted ribosomes are collected into tubes of 4.5ml
?. [Ribosome purification]
Pick the tubes that have the biggest absorbance in wave length 280nm
?. [Column recovery]
Wash with 45ml of milli-Q water
?. [Column recovery]
Wash with 45ml of NaOH (1M)
?. [Column recovery]
Wash with 45ml of milli-Q water
?. [Column recovery]
Wash with 45ml of Acetic Acid (0.1 M)
?. [Column recovery]
Wash with 45ml of milli-Q water
?. [Column recovery]
Equilibrate with 45ml of Ethanol 20%
After the cleaning the column is stored at 4oC in 20% ethanol and can be reused
?. [Ribosome collection]
Add 14μl of β-Mercaethanol to 28ml of Cushion buffer and mix
We will use two tubes for Ultracentrifuge
?. Place 14ml of Cushion Buffer to each of the Ultracentrifuge tubes
?. Carefully place the ribosomes solution on top of the cushion buffer and make sure that we still see the separation layer
?. Ultracentrifuge for 16 hours with 24000RPM (100000RCF) at 4oC
?. Discard the liquid
The pellet of ribosomes might not be visible at this point
?. Add 2μl of β-Mercaethanol to 4ml of Ribosome Buffer and mix
?. Add 500μl of Ribosome buffer to the wall of the tube and slightly spin to wash the cushion buffer on each tube and then discard the liquid
?. Repeat step No 39
?. Add 100μl of Ribosome buffer to each tube and resuspend the cells with a magnetic stirrer on ice for 10min at 200rpm
?. Collect the samples to a single tube ( Tube No1)
?. Wash the centrifuge tubes with 100μl of Ribosome Buffer and add that to another tube (Tube No2)
?. [Measurements and concentration]
Load the solution to a 0.5mL Amicon Ultra filter with 3kDa molecular weight cutoff and centrifuge at 14000RCF for 10min at 4
Use two separate filter for Tube No1 and No2
?. [Measurements and concentration]
Dilute 1μl of the concentrated solution to 99μl of ribosome buffer (or milli-Q water?) and measure the absorbance at 260nm
?. [Measurements and concentration]
Optionally , if the absorbance of Tube No2 is more than 10% of the one measured for the Tube No1 you can mix the two samples and repeat steps 44 and 45
?. [Measurements and concentration]
Adjust the final concentration 10uM by dilution with ribosome buffer
Absorbance of 10 corresponds to 23uM concentration
?. [Storage ]
Aliquote the final solution according to your needs (0.9μl are needed for one 5μl reaction) and store at -80oC
?. [Ribosome purification]
Equilibrate columns with 60 ml buffer C