Swartz:Protocols/Ribosome purification

See https://docs.google.com/document/d/1NIr0ldJ3qRHlnsK1kKO-WOEMJor2i8oIsP89cbREMxE/edit

Ribosome Purification

Buffers: Buffer A

20 mM Tris HCL pH 7.2

100 mM NH_4Cl

10 mM MgCl_2

0.5 mM EDTA

6 mM BME

Buffer B:

20 mM Tris HCL pH 7.2

100 mM NH_4Cl

10 mM MgCl_2

0.5 mM EDTA

6 mM BME

37.7% Sucrose

Buffer C:

10 mM Tris OAc pH 7.5

60 mM NH_4Cl

5mM Mg(OAc)_2

0.5 mM EDTA

6 mM BME

BME should be added just before use -it may be helpful to make 5x stocks of each buffer without BME (and sucrose for Buffer B) and prepare 1x buffers just before use

Day 1:

Resuspend frozen cells in Buffer A at 1g/mL

-typically use 10g cells in 10mL Buffer A

-cells should be from an E. coli fermentation, preferably harvested while still in exponential phase

-the hand homogenizer can be used for resuspension

Add ~150U of RNAse inhibitor (from Invitrogen, usually at 10-30U/uL)

Lyse by flowing through homogenizer 1 pass at >20,000psi

Spin down lysate at max speed (about 12,000 rpm) for 15min in Sorval RC5B centrifuge in 30mL centrifuge tube

Pour supernatant into fresh 30mL tube

-add 2-3mL Buffer A to pellet and add this to fresh tube as well to remove soft top layer

Spin again for 15min at ~12,000rpm

Collect supernatant by pouring into fresh Falcon tube

During spins, prepare Buffer B

Add 3mL Buffer B to 8 different Type 65 rotor ultracentrifuge tubes

Pipette ~2-2.5mL of lysate supernatant to each ultracentrifuge tube being careful not to mix

-there should be visible separation between the 2 solutions

Mass tubes pairwise to <5mg

Spin in Beckman L8 80M ultracentrifuge with Type 65 rotor at 4°C, 50,000rpm, 20 hours

-it is recommended to pre-cool the rotor to 4°C before use

Day 2:

When ultracentrifugation is complete, there should be a brown ring about halfway up each tube

Carefully pour off supernatants

-ribosomes should form a clear, soft pellet on the bottom of the tubes

Invert tubes and place at 4°C to dry for about 10min being careful not to lose pellets

Add 250uL Buffer C to each tube

Rock samples at 4°C for 3-4 hours to resuspend

-cover tubes with parafilm

Determining ribosome concentration:

Measure A280 and A260 using spectrophotometer

-Dilute samples approximately 1000x for measurement

-A260/A280 should be just under 2

To calculate concentrations, assume 15 A260 units = 1mg/mL of ribosomes

-E. coli ribosome MW is 2.7 million Daltons