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A small repository of synaptic protein information

Caution! In converting these protocols to HTML, some units have been scrambled.
Specifically, microliters (ul) often appear as milliliters (ml). Think first!

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1. Pour 1.5ml of the culture into a microfuge tube and pellet 5K 3-4'. Aspirate off the medium and resuspend the cells well in 100ul ice-cold Solution I. Vortex gently to ensure complete resuspention.
2. Add 200ml freshly prepared Solution II and mix by inverting the tubes several times (DO NOT VORTEX); store tubes on ice 5' or more.
3. Add 150ml ice-cold Solution III and vortex gently. Allow to sit at least 10'.
4. Centrifuge 5' @ 4oC and transfer sup to a new tube.
5. Add RNase A to 20ng/l (4ml of 2mg/ml stock) and digest 4' @ 37oC.
6. Phenol-chloroform extract 1X and ppt with 2vol EtOH (no salt). Let the tubes stand 2' @ RT and then spin 5' @ 4oC. Wash pellet with 70% EtOH and vacuum dry.
7. Resuspend in 50ml TE and store @ -20oC.

Solution I
50mM Glucose 4.5g
25mM Tris, pH 8 6.25ml 2M
10mM EDTA 10.0ml 0.5M ddH2O to 500ml

Solution II
0.2N NaOH 10.0ml 10N or 100ml 2M NaOH
1.0% SDS 25ml 20% 100ul 10% SDS
ddH2O to 500ml 800ul H2O to 1ml

Solution III
3M K 147g KAc
5M acetate 68ml glacial acetic aci dddH2O to 500ml

For 10ml preps:
1. Pellet cells for 10' @ 4krpm, resuspend in 200ml Solution I and transfer to mfuge tube.
2. Add 400ml Solution II, mix by inverting and place on ice.
3. Add 300ml ice-cold Solution III, vortex gently and inc. 3-5' on ice.
4. Spin 5' @ 4oC.
5. Transfer 600ml to a new tube, RNase digest, phenol-chloroform extract 1X and ppt. with 600ml isopropanol. Pellet 5' @ 4oC and wash with 70% EtOH.
6. Resuspend in 250-500ml TE.
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