1. Purification of Tight coupled ribosome.

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Lysis buffers : Buffer A (20 mM Tris–HCl (pH 7.4), 100 mM NH4Cl, 10.5 mM Mg(CH3COO)2, 0.5 mM EDTA, and 1 mM DTT) + 0.5 mg/ml Lysozyme, 40 U RNase free DNase, 1 mM phenylmethanesulfonyl fluoride and RNaseOUT (Invitrogen)

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High salt sucrose buffer: Buffer B (20 mM Tris–HCl (pH 7.4), 1 M NH4Cl, 1.1 M sucrose, 10.5 mM Mg(CH3COO)2, 0.5 mM EDTA and 1 mM DTT).

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Base buffer for linear sucrose density gradient: Buffer E (20 mM Tris–HCl, (pH 7.4), 100 mM NH4Cl, 10.5 mM Mg (CH3COO)2, 0.5mM EDTA and 1 mM DTT) + Vary this buffer with 5 to 40% sucrose (w/v) to layer for density gradients. In case you use a gradient maker, you may need only 0 and 50% stock. 

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Preferred volumes for ultracentrifugation rotors and tubes:

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Fixed angle

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Preparative runs 3.4 ml, 13 ml or 33/40 ml rotors. This is highly dependent on the amount of cells you are processing. 

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Swing buckets

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Preparative runs 13 ml or 33/40 ml rotors.

Analytical runs 2.2 ml to 12 ml tubes 

 

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I have used them from Thermo and Beckmann. In my opinion, it does not matter what you use; only rotor names and fitting tubes will change. 

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A rough outline for a prep :

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Day 1:

-Harvest cells from exponential phase. Note this may change on very special situations when you intend to analyse ribosomes from another growth phase. 

-Resuspend frozen cells in Buffer A at 1g/mL. of buffer A with additives

-Lyse by flowing through french press 3 pass at >15 kpsi or Freeze thaw lysis. Cryo pulverization also works very nicely. Lately that has become my favourite. 

-Spin down lysate at max speed (about 30,000g) for 30min at 4 degree C. Use Oakridge kind tubes

-Pour supernatant into fresh 50mL tube

-Collect supernatant by pouring into fresh Falcon tube

-During spins, prepare Buffer B with Ultracentifuge tubes

-Pipette equal volumes of laysate over buffer B in Ultracentifuge tubes

-there should be visible separation between the 2 solutions

-Mass tubes pairwise to <5mg. Never forget this. 

Spin in desired ultracentrifuge at 4°C, 100,000rpm, 3 hours

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

-When ultracentrifugation is complete, the lysate may have diffused into the next layer.

-Carefully pour off supernatants

-Ribosomes will 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. Preferably in a cold room. 

-Add Buffer A (without additives) to each tube

-Rock samples at 4°C for 3-4 hours to resuspend with a cover at top.

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Determining ribosome concentration:

-Measure A280 and A260 using spectrophotometer. Note not to use Bme as a reducing agent as it absorbs at 260 nm.

-Dilute samples to  100x and 1000x for measurement and final A260/A280 should be just under 2. This is good estimate of purity but an rRNA extraction and SDS PAGE for proteins is reccomended. 

 

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

-E. coli ribosome MW is 2.7 million Daltons

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