Cell Lysis - (Aug/03/2006 )
I am purifying my protein through affintity column and for cell lysis i am using NP10 buffer.
Fisrt i add lysozyme 0.2mg/ml and kepp it on ice or room temerature for 1 hr. After this step I add Triton x-100. Problem is after lysozymes step a lot of cell mass settle down and its too vicsous and it did not dissole again upon skaing and it form long thread like mass when i keep it at rom temperature. if i do lysis on ice then triton x-100 did not dissolve in the lysis mixture and it aggregated like beads with cell debirs at the bottom.
what is the cell mass. Either protein is precepitating or some thing else.
regards
Fisrt i add lysozyme 0.2mg/ml and kepp it on ice or room temerature for 1 hr. After this step I add Triton x-100. Problem is after lysozymes step a lot of cell mass settle down and its too vicsous and it did not dissole again upon skaing and it form long thread like mass when i keep it at rom temperature. if i do lysis on ice then triton x-100 did not dissolve in the lysis mixture and it aggregated like beads with cell debirs at the bottom.
what is the cell mass. Either protein is precepitating or some thing else.
regards
Hi Samita...
Can you explain NP-10 buffer...is this nonidet P-40 diluted? My lysozyme is kept at 20 mg/mL stock and I use it from frozen stock at 200 ug/mL of lysis.
Here's what I do...add 2 mLs of lysis buffer (of your choice best for your protein) to frozen cell mass. For four litres of culture, I usually end up with about 18 to 25 mg/mL depending on protein and plasmid and cells. The cell paste will be frozen overnight (usually). Then thaw in your buffer of choice and add 200ug/mL of lysozyme (final concentration). Let sit on ice for 20 minutes. Should you decide to add additional detergent, add it after lysozyme digestion. Then I sonicate 6 times 30 second intervals with 30 second rests to keep cold on ice. After that, I will spin at 23K rpm for 30 minutes at 4 degrees C.
Then, I add the cleared supernatant (if soluble protein) to a column, filtered with 0.2um syringe if necessary.
You mention your viscosity. If this is a problem, maybe your are letting your cells lyse too long and the viscosity is usually due to nucleic acid. Sonication will solve this. Also if you have no sonifying machine, use a syringe with a 18 guage (spelling?) needle and draw the culture through it to shear the nucleic acid threads.
Then, once the protein is cleared and soluble, I will load my column on FPLC.
Best Wishes,
Cindy
Gloopy mess is genomic DNA alright. Try adding a bit of DNase to your lysis buffer while you do the lysozyme step. When the lysate is no longer viscous, you're right to continue.
Typically, I do 2 or 3 cycles of freeze-thaw, with liquid N2 for the freeze step.