Preparation of crude extract
| - Protein crystallography
Main steps:- Protein purification
- Equipment & Reagents
- Stock solutions
- Chromatographic columns
- Crude extract
- pre Chromatographic step
- Chromatographic step
- Common sense
- Charts & Tables
Special:- Programs for crystallography
- X-ray detectors
Basic tutorials:- Chemistry
- Amino Acids
Xtal community:- CCP4BB
Disruption of bacterial cellsMethods for cell disruption can be divided into two groups:
For full release of the soluble cytoplasmic proteins a combination of non-mechanical and mechanical methods should be employed. For example it is widely recommended to apply lysozyme treatment followed by ultrasonication. I've found a combination of using osmotic shock, freezing-thawing and sonication simpler and quicker.
For effective sonication, cell suspensia has to be prepared by adding 8-20 ml of buffer per gram (or ml) of cell pellets. During sonication cell suspensia should be placed in an ice bath.
To prevent the protein denaturing due to the heat released by the probe, sonication should be performed in 3/4 cycles for 20-25 sec at 16-micron amplitude (the maximum on a Soniprep 150 machine), allowing the sample to cool between sonication sessions.
Tip: To save time on this step it is useful to divide cell suspension into 3-4 portions and treat the portions one after another thereby allowing each portion to cool while you treat the other portions. Also, you can add small peaces of ice in to cell suspension between the sonication sessions.
Removal of cell debris (insoluble part of cells)Practically the only method used for the removal of debris is centrifugation.
A method called differential centrifugation can be applied to separate a homogenates components by size.
Normally to purify soluble cytoplasmic proteins we do not need to remove ribosomes or all the membranes from the crude extract, we only need to remove big particles (bigger than 10µm, which is the mesh size of the net used in a chromatographic column adaptor).
Empirically the optimal protocol has been found to be as follows:
For purification of membrane proteins you only need to spin down the large pieces (this is done by centrifugation at 10000g for 10 minutes) and then membranes need to be collected, this is done by ultracentrifuge at 100000g for 1 hour.