Greetings!
I am currently working with a kinase name Microtubule affinity regulating kinase 2( MARK2). Every time I purified this protein, I have never got a good yield. Usually after purification, the concentration of the protein is ~1uM (with volume of 400 uL) and none of my assay work with this concentration. The highest concentration I have ever got is 5 uM.
Here is the buffer I am using for it:
Binding Buffer (50mM Tris, 200mM NaCl, 50 mM Imidazole, 5 mM 3-[3-cholamidopropyle dimethylammino] propanesulfonate, 2mM benzamidine hydrochloride, 1 mM beta mercaptoethanol, 1mM phenylmethylsulfonyl fluoride)
Elusion Buffer ( Same as binding buffer except imidazole concentration is 500mM)
Dialysis Buffer (50mM Tris, 5mM Mgcl2, 2mM EGTA, 0.5 mM Benzamidine HCL, 0.5 mM phenylmethylsulfonyl fluoride)
Very recently, I just tried to optimize it by following way:
Bacterial cells were grown untill the O.D value reaches at 0.8 and 1L LB media containing the MARK2 expressed cell were centrifuged into one bottle.
The imidazole concentration in biding buffer decreased to 5mM
I have used a gradient elusion for elusion buffer which is, from elusion 1 to elusion 5, I have gradually increased the concentration of imidazole to 100mM to 500mM
I have also incubated the Ni-NTA column for 2 hour with the supernatant of bacterial cell after spining down.
I have really seen a very good band of protein when I run it on the SDS-PAGE. I have attached the gel image here for your reference (MW of the protein is 87kd). However, I do not understand what happened to my protein when I tried to concentrate it. the concentration went up to 26 uM. And then, when I took out all the protein from the Amicon filter unit and did the final centrifugation, the final concentration was 1.2uM again. I just do not understand what is happening right here at the end. Can you please suggest me a way or any idea that can help me to concentrate my protein with proper yield??
Thanks in advance.
Dear Khairun,
As I can see from your gel you dont have good induction of the protein, to overcome that, may be the codon optimization is required. I conclude this from many in specific bonds on your gel.
Further, keep in mind that many his tagged proteins tend to aggregation. May be you get this during concentration. Are your spin columns with PES filter? Others can stick the protein
Sometimes the yield (or the concentration of protein) can be increased by following. You should prepare 25x concentrated cell extract in wash buffer, lets say 80 ml from from 2 l of culture. Then you drop the NiNTA suspension directly into cell extract and mix gently for 30 min. Let the resin to precipitate and remove the liquid. Add wash buffer and move the resin into the gravity flow column and continue normal purification. In such a way you will decrease the contamination of the protein.
Hey,
I can see the nice band in your gradient eluted sample on SDS gel. For the further optimization of your samples, you should also try to play with pH of buffers, during purification process. I had this problem of low concentration of my protein/enzyme and further it was also not showing good activity. I worked out and set the different pH of my buffers during purification. Also to add more, it is small thing to notice but the pH of your LB media, can also affect the yield of protein. Sometime the different batch of yeast extract varies the pH.
Finally, I would recommend you to speed up the process of concentrating the protein samples. You can divide your sample (if dealing with bulk sample) and also you should take care of using the optimal temperature for your centrifuge. Long time during concentration process, will lead to non-specific binding of protein with the membrane material.
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