Hi all, I was wondering why there is a need for lysis buffer to break the cell and binding buffer for the column affinity chromatography purification?
I'm using sonification to break the cell. Do I really need to use lysis buffer or I can just use equilibrium buffer (0.1M phosphate buffer + 300mM NaCl) for the sonification?
After breaking the cell, I need to purify the protein. Some people told me they use binding buffer. What is the important of binding buffer in purification? Is that it really a need? I had purified my protein with ~50mL washing buffer (0.1M phosphate buffer+ 300mM NaCl+10mM imidazole) and then elute it out with elution buffer. However, there is still a lot of impurities which I suspect that 50mL is not enough for washing. What is you opinion?
Based on your use of imidazol i guess you are using a Ni-Affinity Column. But lets get to the point. Normally if i do his-tag purification my lysis buffer contains some protease inhibitors and lysozym to prevent my desired protein to be eaten by all the protease and second the lysozym to help cracking those little bastards and releasing all that precious protein. Of course that stuff isn't present in my binding buffer.
For your problem with impurity...i guess washing a bit more can't hurt but i would rather raise the imidazole concentration of your washing buffer up to 40 mM (maybe even more...try!) because all this nasty proteins that the bacteria produces itself might contain a few histidine an they also glue to the media...so increasing the imidazol concentration should decrease the impurity. ( I actually also put it directly in my lysis buffer to get those nasty impuritys right off)
But the best way to get a nice single protein would be to run a gradient of imidazole over the column use an FPLC to automatize the purification and boost your result. You are going to love the over night purification and have a nice sleep while getting your protein purified by those fabolous machines!
Hi Thomas, I was thinking of increasing the imidazole concentration. But how can I really sure the eluent doesn't contain my protein at all? My protein size is 99kDa without his-tag size. I'm afraid that higher imidazole will slightly remove my protein at the same time with the impurities. Btw, I'm using manually column purification, with the aid of the pressure pump.
I am afraid the right ammount of imidazole to elute your protein is something you have to try out. maybe you can try a step-gradient and increase the concentration of imidazole by 5-10 mM per step.
Do bradford tests to determine when your protein is eluted
You can run each fraction on SDS-PAGE to see when your protein elutes.
Hi Thomas, I know that Bradford can determine the presence of protein. But how can I know that the protein in the flow through is actually my protein or just the impurities and unwanted protein (like the cell itself produce the protein as well)?
Hi Adam, SDS-PAGE can be a way, but that's too time consuming. To properly running a gel, staining and destaining require at least an overnight. It's quite impossible to leave the protein in the column without elute them out. After the elution, it might need an overnight day to do the dialysis again before for the second purification with slightly higher concentration of imidazole (or else the protein will be washed out with higher imidazole content than the washing solution). Any others better way and solution?
Hi Alex,
You can speed up SDS-PAGE considerably by using mini-gels. The pre-cast NuPAGE mini-gels from Novex with MES running buffer take 35 minutes to run. They can be stained in a few minutes with Expedeon Instant Blue colloidal Coomassie Blue stain, which requires no washing, fixation, or destaining. The whole process from sample prep to stained gel can be done in an hour or so.
Have you tried a quick Bradford? Its quick and dirty but could be useful for your application. Use a microwell plate and mix 20 µl of each fraction with 200 µl of Bradford reagent. The intensity of the blue should be very high when your eluted protein of interest is present, taking in account that the protein of interest is the main protein in your elution and in high concentration
Hi Thomas, I did do the Bradford test. The Bradford assay can tell me how much is my protein concentration, but how can it differentiate between my protein and the impurities (unwanted protein)? I think I'm going to increase the imidazole of my wash buffer from 10mM to 30mM. I hope this will help. And thanks for your help!
Well if you are using a recombinant protein with an induced expression you simply have to guess by the amount of protein which fraction contains your protein otherwise you have to use SDS-PAGE.
If you are using a step-gradient for example elution first with 50 mM Imidazole, than 100 mM Imidazole and 200 mM Imidazole etc. considering that the main protein binding to your affinity resin should be your protein of interest the really blue ones should be your protein.
I was thinking if I'm directly using step gradient elution for purification, without the washing step, then run the sds-page to view the presence of my protein. I will start with lower concentration like 10mM, followed by 20mM, 30mM, 40mM and so on. In this case, I know which concentration will elute out my protein, and I can reload the sample for 2nd purification with lower imidazole concentration than the one before for the washing step. Or perhaps I can directly get the pure protein after the step-gradient elution. Do you think this will work? Will it sound a bit weird?
I dont think you should skip the washing step. Because the washing step is there to elute non-binding proteins and the gradient is there to elute non-specific binding proteins. The rest sounds ok. Good luck!
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