I am using E.coli BL21 Plyss for a recombinant protein production. The cells debris becomes too sticky after lysis. I spin down the pellet at 13000 g (that is the maximum available here). The problem is I could recover only 45-50% of the supernatant. What can i do to recover most of the supernatant?
Two problems can occur:
- the total volume (fluid + pellet) in the tube is reduced during centrifugation, that is, part of the sample leaves the tube and is spilled into the centrifuge. That shouldn't happen, it causes a biohazard and leads to rotor imbalance, which damages the drive bearing. Have somebody show you the proper technique. Also, inform the safety officer, who will instruct you on the cleaning and disinfecting required. Note: failing to report such mishaps asap will really get you in a soup!
- The tubes are as full as they were when you started the centrifugation. Then the question is why you can't recover half of the supernatant. Is the pellet stirred up easily? In general, I find it easiest to slowly pipet the supernatant of the pellet.
Engelbert Buxbaum The tubes are full, but the pellet comes along with the supernatant and doesn't settle down. I can remove only 45% of supernatant without disturbing the pellet. Is there any other way where i can recover most of the supernatant without disturbing the pellet
DEar SOndarya
2 questions:
1) How long did you centrifuge at 13000rpm?
2) Which buffer are you using for the lysis?
3) Which biomass/buffer ratio?
generally i'm using about 10ml/g of biomass of my buffer (tris 20mM, imidazole 10mM, NaCl300mM ph=8) and after sonication and a centrifugation of about 30 minutes at about 13000g and 4°C' i'm able to well separate pellet and surnatant.
Generally at these speed the surnatant still contains some debries that can block the coloumns and therefore to avoid it i perform sample filtration at 0,22uM before load it into the IMAC coloumn.
best regards
Manuele
Try adding incremental amounts of NaCl. It should reduce viscosity and allow recovery of more supernatant.
Manuele Martinelli 1. I'm centrifuging for 60 minutes in 13000 g at 4°C
2. I use PIPES pH 6.8 for lysis
3. The biomass buffer ratio is 15 ml/g of biomass
Sebastian Schmitt
Thanks for your suggestion. By redissolving the pellet I was able to recover most of the protein.- Badges
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