Preparation of samples for analysis of solubility of expression

"Bug buster" method

1. Take one tube of reagent B from the freezer and add 0.1 ml of reagent A (stored at room temp).
2. Put about 20 microliters of cell paste pellet into a tube; to get this, spin down 1-1.5 ml of culture or take a bit of frozen cell paste.
3. Add 0.1 ml of reagent A+B to the cell paste. Suspend it properly by rod.
4. Place on a rotating platform and incubate for 10 min.
5. Spin down debris at 17000rpm for 5-10 min in the refrigerated centrifuge.
6. Take out supernatant and place into another tube. Mark it 'S'.
7. Add 0.1 ml of 4% SDS solution to the tube with pellet and suspend it properly with rod. Mark it 'P'.

1. Suspend cell paste from 50 ml culture in 2-3 ml of 50mM tris-HCl buffer pH 8. Place into a 5 ml plastic (universal) container. Sonicate on ice with the medium probe. Make 3 cycles for 6-7 sec. at maximum power. Allow sample to cool between cycles. Add small pieces of ice in to the container for better cooling.
2. 1.Take 1 ml of homogenate and spin down debris for 3-4 min at 13000 rpm in the bench top centrifuge or, better still, in the refrigerated centrifuge at 4°C, for 10 min, at 17000 rpm.
3. Separate supernatant fraction into another tube. Mark it 'S'.
4. Re-suspend pellet in 1ml of water or buffer or 2% SDS. Mark it 'P'.
5. Alternative way. Suspend pellet in 0.1 ml of 50 mM tris pH 8.0, 1M NaCl, spin down pellet as above. Separate supernatant fraction in to clean tube. Mark tube S1. Go to step 4.
6. Check protein concentration in supernatant fraction (tube S) and in tube S1.
7. To prepare a "Soluble proteins" sample take about 20 µg of total protein from tube S for 1 sample for gel, add water to make total volume 15µl if necessary. Take some 10 µg of protein or so from tube S1 if there is any significant protein concentration.
8. To prepare "Insoluble proteins" sample take the same volume from the tube P as you took from tube S for 1 sample for gel, add extra SDS (10% solution) so as to make the SDS concentration about 4%.
9. Add 4x-Sample Buffer and 10x-Reducing agent to the both samples and boil them for 2 min. Apply on a gel.