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.

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