I am trying to purify Cas9 and T7 for my experiment but I am not being able to get purified proteins. For pre-assy, I used PBS 1x buffer to wash pellets, lysozyme and DNAse I used, sonication for 15 minutes on ice water, 0.1 % trition used and ice incubation for 15 mintues, centrifuged @6000 rpm 4°C, Ni-NTA beads on supernatant, 1 hr incubation on ice, washed with PBS 1x buffer 3 times, elution with 500mM imidazole. When loaded on SDS gel, I do not find the corresponding band. Can you please suggest effective ways to carry out this pre-assy ? thanks
Did you check the insoluble fraction after lysis? I don't know about T7 but Cas9 is known to form inclusion bodies unless produced at low temperatures.
Hope it helps!
Hi Alejandro,
I used a control after lysis before using NI-NTA beads. In the control sample, I can see bands with degradation. While, after the purification, the band simply disappears. I am not sure whether my purification steps is not working or it's simply not working with PBS. To note, I induced the expression of protein at 30°C with IPTG. Do you have any suggestions for me. Many thanks.
You have to analyze fractions of the entire purification process to see where the protein is going. The sonicated lysate, the soluble fraction, the insoluble fraction, the fraction that did not bind to Ni-NTA, the Ni-NTA eluate and if possible, what comes out of the Ni-NTA beads after treating them with acid GuHCl. Then you can take proper action depending on the results. Also, Western blotting of these fractions with antibodies against the protein and against the tag will give you valuable info (such as e.g. is the observed protein degradation removing the tag? Is the protein not binding the resin despite the tag being present? All these scenarios have different solutions). It does seem like a lot of work but you can do that in a couple of days, and will save you a lot of time down the road.
Also, in the future, posting a picture of the gel will help you get meaningful answers. Oftentimes the fresh eyes of outsiders are able to spot details we might have missed.
Hope it helps!
And make sure that you do not denature your samples during sonication. You write that you did it on ice-water. Make sure to pulse the sonicator with sufficient pauses between pulses. Also, 15min sounds awfully long; a third of it should be more than sufficient.
The cavitation bubbles, which are produced during the sonication process, can get very hot. Temperatures as high as 5500K have been reported - this is the same temperature as on the surface of the sun! This heat has to be controlled and given time to dissipate. or you end up cooking your protein.
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