I'm looking for a lysis buffer recipe and protocol for lysing an E. coli strain. The background is that I will be using IPTG to induce the expression of a plasmid with a specific cDNA. I ultimately want to run the bacterial cell lysates on an SDS-PAGE gel to look for the expression of the cDNA (now protein). I found a recipe from Cold Spring Harbor, but it lacks specific instructions on its use.
These are my conditions to lyze and purify a recombinant protein in non denaturing conditions. Do you have an idea whether the protein will be soluble or not (if not, consider using 6M guanidine for lysis and purification)
Protein purification:
Prepare the buffers for lysis, wash and elution as follows and keep at 4C.
Base : 450ml of 50Mm NaH2PO4, 300Mm NaCl (3.45g and 8.77g respectively), adjust the pH to 8. (Make the double and keep the remaining)
Split the volume as below:
Lysis buffer (10mM Imidazole): 117ml Base + 88.5mg Imidazole (qsp 130ml H20)
Wash Buffer (20mM Imidazole): 288ml base + 435.7mg Imidazole (qsp 320ml H20)
Wash Buffer 2 (50mM Imidazole) : 45ml Base + 170.2mg Imidazole (qsp 50ml H20)
Elution Buffer (250mM Imidazole) : 45ml Base + 851mg Imidazole (qsp 50ml H20)
Filter sterilize all buffers.
-lyze the pellet in 30ml Lysis buffer (1/10 culture volume) + 94mg lysozyme. Incubation 30’ on ice
- Sonicate on ice: 2 sec pluse x 30 times or 5 sec pulse 8 times.
- Clear lysate : spin for 30’ at 10000g (SW34 rotor) and save the sup
- Apply sup to Nickel agarose beads (washed 3 times in Wash Buffer 1: rocker for 5’ then spin at 4000rpm). Incubate at 4C for 1h. Spin and save flow through
- 3 wash with Wash Buffer 1 and then 1 wash with Wash Buffer 2
- Add to the beads 10ml of elution buffer and transfer to a column
- Collect 1ml fractions and analyse on SDS-PAGE
you can add a protease inhibitor (Roche tablets) if the downstream steps permits it.
you can try lysozyme e.g. from Roche? If you use 6M guanidine it is defintely denatured.
Why not boil them in 2xLaemmli buffer before putting them on SDS-PAGE as you would anyway before loading your gel? It works good in PCR to boil E.coli directly in the cycler for 10 min before you do your run. (I never tried for PAGE it it is just a thought!!!)
The method described by Sam Manna works really well, I have used it with big pellets from 5L bacterial cultures for protein isolation. No additional enzymes, 50 ml Falcon vial with bacteria suspended in phosphate buffer, some liquid nitrogen, water bath (I was thawing at 37 centigrades with shaking). After 4 cycles almost nothing was still alive (I have checked by seeding on bacterial plates - I had some single colonies only). Very simple, easy, cheap and really efficient. If You are afraid about Your protein You can add some DTT - it is not very influenced by freezing and thawing as it is not a protein just small reducing molecule.
put the bacteria in a simple kitchen Microwave machine and exposes for 1 min.
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