Hello, can you help me ?
I'm working with E.coli BL21 transformed with a plasmid that codes for a 13.271 kDa His-tagged periplasmic protein.
I've already done the induction part with IPTG, but I'd like to verify the correct expression using a Western blot. The problem is that I'm finding a lot of contradictory information and in the end, I don't know which parameters would work best in my case.
For the moment, I've written this protocol for lysis and extraction only :
Washing: Place samples on ice and resuspend the pellets in PBS (previously cooled to 4°C) by vortexing. Centrifuge for 1 minute at 4°C and 4150g and discard the supernatant.
Resuspend the pellets in cold lysis buffer (5 ml per gram of cells).
Stock solution PMSF (X100)
PMSF 100 mM
Ethanol 100% qsp 10 ml
Lysis buffer (1X)
MgCl2 1 mM
Lysozyme 10 mg/ml
PMSF 1 mM
DNAse 20 mg/ml
PBS pH 7,4 qsp 40 ml
Add lysozyme and PMSF (= anti-protease) just before the experiment. Add DNAse after sonication.
Lysozyme has an Mw of 15 kDa. If the protein being studied has a similar Mw, it may be better to use a different lysis buffer. Do not add EDTA if the protein of interest has a histidine tag.
Make a stock solution of PMSF as it is not very miscible in water.
Still on ice, sonicate the samples for 8 minutes at a rate of 30-second cycles every 50 seconds (6 cycles) at a frequency of 23 kHz and an amplitude of 10 microns. The probe must be completely immersed in the sample, without touching the tube.
Centrifuge for 1h30 at 4°C and 4150g. Separate the supernatant from the pellet (a pause of a few days is possible at this stage if the samples are stored at -80°C in glycerol). If the protein of interest is membrane-bound, keep the pellet; if it is cytoplasmic or periplasmic, keep the supernatant.
After 1h30 centrifugation, depending on the protein of interest, add 1X Laemmli buffer to the pellet (how many ml?) or 2X Laemmli buffer to the supernatant.
Stock solution Laemmli buffer (4X)
Tris pH 6.8 200 mM
SDS 8 % (m/v)
Glycerol 40%
Bromophenol Blue 0,4 % (m/v)
DTT 400 mM
H2O qsp 40 ml
Store the loading buffer without DTT at room temperature. Add DTT just before using the buffer. 200 mM β-mercaptoethanol can be used instead of DTT.
Heat the samples for 5 minutes at 95°C. Then cool for 5 minutes on ice.
Keep samples on ice for use (or freeze at -80°C in glycerol for a few days for later use).
Can you tell me what you would change in my protocol please?
And if you have any recommendations for the purification part, I'd be interested.
If you just wish to confirm expression of your protein then there are much simpler ways to do it. You can grow a culture and induce with IPTG (be sure to also include a negative control like vector only without insert). Then after appropriate induction period collect the cells by centrifugation. I think about 100ul of cells in a microfuge tube is plenty. Remove supernatant and resuspend in small volume of buffer and add 2X Laemmli and heat to 95 for 5 min. The cells will lyse and all the proteins will be solubilized. Run sample side by side with negative control to see if you can identify your band.
For larger scale your protocol would generally work fine but there are also easier ways, such with lysis buffers like BPER or similar which are single step lysis and work for many proteins.
Lysis is simple, here is a common protocol, https://wolfson.huji.ac.il/purification/Protocols/InclBodCont.html
With respect to purification, you do not provide enough info, is the protein tagged? is the concentration sufficient? etc.
Which periplasmic translocation tag are you using? Fundamentally, periplasmic extraction can be accomplished via a simple osmotic shock procedure. The osmotic shock supe can then be used for ni-nta resin purification of your protein. If you know it is being expressed in your cells then the rest should follow with appropriate culture. I would also add that baffled flasks greatly improve yield.
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