I'm working with some short protein with size around 20 kDa. I'm expressing the protein in E. coli BL21(DE3) system at 25 degree, 150 rpm in YT media. While I'm checking the proteins in cell lysate using 4-16% Tris Tricine SDS PAGE, I'm getting quite good protein band. However, when I'm growing the same clone in larger volume (600ml) and then after cell lysis (50mM tris, 250 mM NaCl, 5% glecerol, 2mM DTT, lysozyme 0.1mg/ml, PMSF) followed by 30 minute incubation and short sonication (10 second pulse, 3 minute), I'm not getting the specific band on gel. However, upon running the clarified lysate on Bio-Rad Nuvia Ni-NTA column (washing buffer- 25 mM tris, 300 mM NaCl, 40 mM Imidazole, Elution buffer 50 mM tris, 500 mM NaCl, 400 mM Imidazole), I'm getting some protein eluting from the column. However, upon running the protein in 4-16% Tris-Tricine SDS PAGE, no single band was obtained. Instead, a wide size range of protein, similar with cell lysate was obtained. The eluted protein was attempted for dialysis and was found to be stable at high NaCl concentration (400mM) in dialysis buffer. At lower concentration of NaCl, protein got precipitated immediately.
Hi,
I think that the suggestions of Sebastian are highly relevant. I want to clarify the question, your problem is that you don't observe the band of your protein on the SDS-PAGE after Ni-NTA? Can you label bands of the marker and indicate where you expect band of your protein? Did you load also Ni-NTA Flow-Trough sample?
With regards,
Tom
Hi,
According to your description, I feel that your target protein is not very stable. I agree with Sebastian Schmitt that gradient elution may provide you with a relatively pure protein. In addition, it is common to have many protein bands after affinity chromatography only, because Ni-NTA resin may bind to certain proteins non-specifically, thus you need to purify the target protein through further operations such as gel filtration or ion exchange. When purifying large amounts of proteins, please pay attention to controlling the temperature and time to ensure as low temperature and fast as possible.
All the best!
Thank you all for your wise suggestion. I want to mention that the PI of the protein is quite high (PI-11.3). I used a gradient elution in FPLC system. The run summery was as follows.
After sample application, column wash with 5 cv wash buffer as said previously, followed by 6 cv gradient elution and 5 cv elution with elution buffer only with a flow rate of 2ml/min. From my previous experiments, I have observed when adding glycerol, less amount of protein was eluted. The eluted fraction was run in P6 desalting column with 50mM tris, 500mM NaCl and was checked on PAGE. However, similar problem was found. Shall I use a different Ni-NTA column or shall I proceed for gravity based purification or electroelution? I need to retain the biological activity of the protein also.
- Badges
- Science method