I ´purify GST-tagged transcription factors from E.coli (BL21 RIPL).
I either lyse my cells with ultrasound (sonicator) or with pressure (french press).
Sometimes I encounter a turbidity in my lysate after centrifugation (18 000 g, 15 min) - the lysate is still relatively clear, however. I remove potential suspended matter always with a 0.45 µM filter before loading my lysate on the ÄKTA.
Every time I see this turbidity I need significantly more filters (they clog) and get almost no purified protein in the eluate.
What can this turbidity be? How can I avoid it?
Thanks for your help!
Hi Max,
Are you including sufficient DNaseI in your bacterial suspension as release of genomic DNA during sonication can result in high viscosity and enhanced turbidity.
Best
SU
Hi Sandeep,
I don't include DNaseI because I want to perform functional assays with the transcription factor after the purification and some people told me the risk of DNase contamination might be relatively high.
I actually thought of DNA contamination and that was why I went from sonication to french press but the problem reoccurred.
Hi Max, I hope you are well,
I guess that the turbidity you are seeing is due precipitated protein, so have you look for better lysis buffer?, I think you might revise the ionic strength and pH, because a great amount of your sample is precipitating in that conditions and normally happens due to these parameters.
On the other hand, I think you can add DNAse without comprometing the protein homogeinity when you have finished the purification, because, although the DNAse and your transcription factor share the same substrate, the last is GST-tagged. So, you are going to easily separate with an affinity chromatography.
Besides, if you use sonication as a cell disrupting method, you devoid the need of adding DNAse because longer molecules (as DNA) are fragmented in the process and don't apport to the viscosity of the medium anymore.
I hope this can help you, best regards,
Ángel Lobo
Hi Ángel,
I was also thinking about this, however wouldn't it be surprising that this is only occurring sporadically?
My lysis buffer is 500 mM NaCl, 50 mM Tris, pH 7, 20 % Glycerol, 2 mM DTT and 1x Protease Inhibitor.
Best,
Max
Lysate after sonication is often not transparent, but translucent, is it so with you and it should not be turbidity and if still you feel so you can filter it through membrane filter.
First, in order to get rid of turbidity, you could centrifuge your lysate longer and at higher g (e.g. 30 min at 35 000 gmax, i.e. 16500 rpm in an SS34 rotor or equivalent). Don't be too greedy and don't suck up the last bit of supernatant if it apears too viscous, as it will clog the filters. If your protein isn't present in the supernatant, it may be that it forms inclusion bodies present in the pellet. You could check for that by dissolving some of the pellet in SDS-containing sample buffer and running it on a gel (take care of not overloading the gel).
I agree that is is surprising that the turbidity you describe occurs only sporadically. Are you sure that your growth conditions are strictly reproducible ( medium, pH, temperature, tme and temperature of induction etc.)?
The growth conditions should be reproducible in the sense that I use auto-induction medium from the same stocks and use a programmed incubator to regulate temperature. The exact time of induction should be chosen by the bacteria when the run out of glucose.
Could inclusion bodies lead to a turbidity?
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