Swartz:Protocols/Ribosome purification

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m (New page: See https://docs.google.com/document/d/1NIr0ldJ3qRHlnsK1kKO-WOEMJor2i8oIsP89cbREMxE/edit =Ribosome Purification= Buffers: BME should be added just before use -it may be helpful to make ...)
Current revision (16:36, 24 July 2012) (view source)
 
Line 4: Line 4:
Buffers:
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
BME should be added just before use

Current revision

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

Contents

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
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