Moore:Ribosome Purification

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Line 17: Line 17:
===Lysis and Removing Cell Debris===
===Lysis and Removing Cell Debris===
 +
*You can use a pressure cell to shear the cells open.  Works fine for huge preps, but is rather impractical for most volumes.
*You can use a pressure cell to shear the cells open.  Works fine for huge preps, but is rather impractical for most volumes.
*I either use (1) hen egg white lysozyme, or (2) phage λ lysozyme. See: [[Sauer:Lysing_E._coli_with_Lysozymes]].  I generally grow the cells, and initiate lysis, divide the lysate into micro-fuge tubes after the DNase reduces the viscocity, and spin out most of the cell debris (~10 min, full speed), pipette the supernatants to clean tubes, then freeze the aliquots at -80 °C.
*I either use (1) hen egg white lysozyme, or (2) phage λ lysozyme. See: [[Sauer:Lysing_E._coli_with_Lysozymes]].  I generally grow the cells, and initiate lysis, divide the lysate into micro-fuge tubes after the DNase reduces the viscocity, and spin out most of the cell debris (~10 min, full speed), pipette the supernatants to clean tubes, then freeze the aliquots at -80 °C.
Line 49: Line 50:
*After your spin, you will, in most cases, want to discard the supernatant. Aspiration works well, draw from the meniscus and follow the solution down to the pellet.  Suck it dry.  If you did a good job of pre-clearing your lysates, the ribosome pellet will be a crystal clear "lense" that has the consistency of wax.
*After your spin, you will, in most cases, want to discard the supernatant. Aspiration works well, draw from the meniscus and follow the solution down to the pellet.  Suck it dry.  If you did a good job of pre-clearing your lysates, the ribosome pellet will be a crystal clear "lense" that has the consistency of wax.
-
*Add a buffered solution containing magnesium to the pellet.  Don't touch the pellet with your pipette tip, it is really sticky.  I usually resuspend pellets in ~200 μl of buffer.  Place the tubes in a rack and shake then at 4 °C until the ribosomes are in solution. To speed things up, I make mini stir-sticks from pipette tips: put a 200 μl tip on a pipetter, pass it through a flame just until it lights, then blow it out.  The tip will seal shut.  Rinse the tip in and aliquot of ribosome buffer, and eject it in to the centrifuge tube.  Having the strirrer in the tube reduces resuspension time significantly.
+
*Add a buffered solution containing magnesium to the pellet (I use HT-6).  Don't touch the pellet with your pipette tip, it is really sticky.  I usually resuspend pellets in ~200 μl of buffer.  Place the tubes in a rack and shake then at 4 °C until the ribosomes are in solution. To speed things up, I make mini stir-sticks from pipette tips: put a 200 μl tip on a pipetter, pass it through a flame just until it lights, then blow it out.  The tip will seal shut.  Rinse the tip in and aliquot of ribosome buffer, and eject it in to the centrifuge tube.  Having the strirrer in the tube reduces resuspension time significantly.
*When the pellet is gone, transfer the sultion to a clean tube and spin about ten minutes to remove any large bits and membrane contamination.  Transfer the ribosomes to a new tube.
*When the pellet is gone, transfer the sultion to a clean tube and spin about ten minutes to remove any large bits and membrane contamination.  Transfer the ribosomes to a new tube.
-
*Quantify the ribosomes by diluting in a buffer and measure the A<sub>260</sub>. See: [[Sauer:Nucleic_acid_quantitation|Link that has ε for rRNAs]]  
+
*Quantify the ribosomes by diluting in a buffer and measure the A<sub>260</sub>. See: [[Sauer:Nucleic_acid_quantitation|Link that has ε for rRNAs]]
 +
 
 +
*Freeze at -80 °C.
==Tips==
==Tips==
 +
* Most plasticware in the lab binds proteins and nucleic acids.  Avoid using tubes and tips that haven't been tested for binding.  Also, keep a non-ionic detergent in your solutions (I use Tween-20).  I have tested a few brands of tubes for ribosome binding, some are horrible.  I currently use Axygen "Maxymum Recovery" tubes.
* Most plasticware in the lab binds proteins and nucleic acids.  Avoid using tubes and tips that haven't been tested for binding.  Also, keep a non-ionic detergent in your solutions (I use Tween-20).  I have tested a few brands of tubes for ribosome binding, some are horrible.  I currently use Axygen "Maxymum Recovery" tubes.
Line 62: Line 66:
==Buffers and Solutions==
==Buffers and Solutions==
 +
 +
These are solutions I use, other groups use different solutions.  I avoid the use of sodium and chloride: these aren't present in high levels in the cell and NaCl will inhibit translation reactions. High magnesium (5-50 mM)promotes subunit association into 70S particles, low magnesium (~1 mM) allows subunit dissociation.  This process is dependent on the concentration of ribsomes as well, at very low ribosome concentration, the subunits will mostly be dissociated.
 +
 +
===Cell Wash Buffer===
 +
*20-50 mM Buffer, pH ~7.5.  I use 40 mM K<sup>+</sup>-HEPES, pH 7.4.
 +
*Salt.  I use 100 mM NaCl.
 +
*Magnesium to match what's in your lysis buffer.
 +
 +
==="HT" Stock Buffer===
 +
This is a "3X" solution used as a base for a lot of buffers I make.
 +
 +
*300 mM K<sup>+</sup>-glutamate.
 +
*60 mM HEPES-Tris.  Measure for 300 mM HEPES and add Tris base to raise the pH to 7.5.
 +
*0.3 mM EDTA
 +
 +
Filter and store at 4 °C.
 +
 +
==="HT-6"===
 +
 +
*1/3 vol HT Stock Buffer
 +
*6.1 mM Mg-OAc (from 1M stock that has 1 mM EDTA).
 +
*14 mM 2-mercaptoethanol (just use 1/1000 of stock).
 +
*0.05% Tween-20 (from 10% stock solution).
 +
 +
This buffer can grow stuff.  Glutamate is unstable.  Don't use old buffer, keep what you have at 4 or -20 °C.
 +
 +
===10% Sucrose===
 +
 +
Make HT-6, but with 10% sucrose as well.
 +
 +
===Lysis Buffer===
 +
 +
*HT-6 with:
 +
*10 U/ml DNase I (Don't use crappy stuff, use RNase-free, molecular biology grade)
 +
*0.5 mM CaCl (DNase I needs calcium)
 +
*0.1 mg/ml lysozyme (only if using exogenous lysozyme)

Revision as of 15:41, 3 July 2008

Contents

Intro

Ribosomes are large aggregates of RNA and protein. Usually, when you harvest E. coli to get them, most of the ribosomes are in the process of making a protein, so what you end up with are ribosomes with some tRNA, mRNA, and translation factors on them. For some studies, you'll want to find a way to remove these components, see (protocol). In general, they're pretty robust, but you can ruin them if you make some mistakes:

  • Removing the magnesium, either by dilution or using a chelator, will perma-break them.
  • Letting RNAse chew on them. As mentioned below, use a strain lacking RNase I (rna).
  • Heating them over 50 °C, up to and including 50 is fine if the solution is properly buffered.
  • Letting the pH move far away from ~7.5. High pH causes RNA hydrolysis, especially when metal is present.
  • Metal-induced RNA cleavage. Keep iron chelated with a small amount of EDTA.


Protocol

Medium, Growth, and Harvest

  • Early protocols call for growing E. coli in a high-phosphate medium. I usually use LB with 0.2% glucose, they seem to be fine.
  • Rapidly chill the cells on ice when they are in early- to mid-log phase. If you let let the cells cool slowly (like in a cold room or centrifuge) they will cold-adapt and synthesize proteins that shut down translation (not good) (cite). If you want to be serious, you can add ice directly to the culture. Don't use ice from tap water, generally, a frozen buffer of some sort is used, about = volume to the culture. Keep all of your stuff on ice until lysis.
  • Harvest the cells in a 4 °C centrifuge, and resuspend in a washing buffer, re-harvest, then resuspend in a lysis buffer. Make the cells concentrated, resuspending a liter of log-phase bugs in 3-5 mls of lysis buffer works pretty well.


Lysis and Removing Cell Debris

  • You can use a pressure cell to shear the cells open. Works fine for huge preps, but is rather impractical for most volumes.
  • I either use (1) hen egg white lysozyme, or (2) phage λ lysozyme. See: Sauer:Lysing_E._coli_with_Lysozymes. I generally grow the cells, and initiate lysis, divide the lysate into micro-fuge tubes after the DNase reduces the viscocity, and spin out most of the cell debris (~10 min, full speed), pipette the supernatants to clean tubes, then freeze the aliquots at -80 °C.
  • It is a good idea to spin a few times before pelleting the ribosomes in the ultracentrifuge: there will be chunks of membranes that contaminate your ribosome pellet. So, the day you plan to pellet the ribosomes, thaw the lysate aliquots and spin them again, or two more times, each time removing the sup to a clean tube. Keep your stuff on ice.


Making "Crude" Ribosomes by Pelleting Them

Here, the goal is to separate te ribosomes from the majority of other, smaller things in the cell. The lysates are layered onto buffer solutions in centrifuge tubes containing 10% sucrose to make it dense. The lysate will float on the sucrose, but when you spin the solutions under high g-forces, the ribosomes out-run most of the other stuff to the bottom of the tube.

How long do I spin them?

This depends on what centrifuge/buckets you're using, how thick your sucrose layer is, what temp the solution is, and how fast you plan to spin them. You can calculate or look-up the "k-factor" for your rotor (a convenient term from Beckman-Coulter that relates spin time to what you want to pellet).

If you don't know or can't find the k-factor:

k = ln(max/min)/w2 x 10-13/3600

if you know a k-factor, but don't want to spin at speed for which it was calculated:

knew = kcurrent x (speedcurrent/speednew)^2

if you have a k-factor and want to know how long to spin:

t (in hours) = k/S

E. coli ribosomes are 70S, the subunits are 30S and 50S. If you want all of the material to pellet, calculate to pellet 30S.

  • Note: You are spinning through sucrose at 4 °C. This is more viscous than water (for which the k-factor is calculated). You have to increase the spin time because the material moves quite a bit slower through the sucrose. 8% sucrose is about twice as viscous as water (I forget where I read that, I'm getting old). I double the time needed to pellet 30S, or calculate the time needed to pellet 15S (same thing). This does pretty well, most rRNA is in the pellet and most tRNAs and SsrA is in the solution.

The first few times you pellet ribosomes in whatever rotor you choose, check where the material distributes by purifying RNA from each phase and running a gel examples of E. coli RNAs.

Resuspending the Ribosomes

  • After your spin, you will, in most cases, want to discard the supernatant. Aspiration works well, draw from the meniscus and follow the solution down to the pellet. Suck it dry. If you did a good job of pre-clearing your lysates, the ribosome pellet will be a crystal clear "lense" that has the consistency of wax.
  • Add a buffered solution containing magnesium to the pellet (I use HT-6). Don't touch the pellet with your pipette tip, it is really sticky. I usually resuspend pellets in ~200 μl of buffer. Place the tubes in a rack and shake then at 4 °C until the ribosomes are in solution. To speed things up, I make mini stir-sticks from pipette tips: put a 200 μl tip on a pipetter, pass it through a flame just until it lights, then blow it out. The tip will seal shut. Rinse the tip in and aliquot of ribosome buffer, and eject it in to the centrifuge tube. Having the strirrer in the tube reduces resuspension time significantly.
  • When the pellet is gone, transfer the sultion to a clean tube and spin about ten minutes to remove any large bits and membrane contamination. Transfer the ribosomes to a new tube.
  • Freeze at -80 °C.


Tips

  • Most plasticware in the lab binds proteins and nucleic acids. Avoid using tubes and tips that haven't been tested for binding. Also, keep a non-ionic detergent in your solutions (I use Tween-20). I have tested a few brands of tubes for ribosome binding, some are horrible. I currently use Axygen "Maxymum Recovery" tubes.
  • Make sure you don't touch anything that touches your buffers, tips, tubes, etc. with bare hands, directly, or indirectly. You WILL introduce RNase that will degrade your stuff.

Buffers and Solutions

These are solutions I use, other groups use different solutions. I avoid the use of sodium and chloride: these aren't present in high levels in the cell and NaCl will inhibit translation reactions. High magnesium (5-50 mM)promotes subunit association into 70S particles, low magnesium (~1 mM) allows subunit dissociation. This process is dependent on the concentration of ribsomes as well, at very low ribosome concentration, the subunits will mostly be dissociated.

Cell Wash Buffer

  • 20-50 mM Buffer, pH ~7.5. I use 40 mM K+-HEPES, pH 7.4.
  • Salt. I use 100 mM NaCl.
  • Magnesium to match what's in your lysis buffer.

"HT" Stock Buffer

This is a "3X" solution used as a base for a lot of buffers I make.

  • 300 mM K+-glutamate.
  • 60 mM HEPES-Tris. Measure for 300 mM HEPES and add Tris base to raise the pH to 7.5.
  • 0.3 mM EDTA

Filter and store at 4 °C.

"HT-6"

  • 1/3 vol HT Stock Buffer
  • 6.1 mM Mg-OAc (from 1M stock that has 1 mM EDTA).
  • 14 mM 2-mercaptoethanol (just use 1/1000 of stock).
  • 0.05% Tween-20 (from 10% stock solution).

This buffer can grow stuff. Glutamate is unstable. Don't use old buffer, keep what you have at 4 or -20 °C.

10% Sucrose

Make HT-6, but with 10% sucrose as well.

Lysis Buffer

  • HT-6 with:
  • 10 U/ml DNase I (Don't use crappy stuff, use RNase-free, molecular biology grade)
  • 0.5 mM CaCl (DNase I needs calcium)
  • 0.1 mg/ml lysozyme (only if using exogenous lysozyme)
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