Ni_NTA Spin Columns
LB medium
Kanamycin stock solution
Ampicillin stock solution
IPTG stock solution
Buffers A–D
5× SDS-PAGE sample buffer
1. Pick single colonies of transformants into 1.5 ml of culture media containing both ampicillin (100 mg/ml) and kanamycin (25 mg/ml). Also inoculate one 1.5-ml culture with a colony transformed with the control plasmid pQE-40, which expresses 6xHis-tagged DHFR or another appropriate control. Inoculate one extra culture to serve as a noninduced control. Grow the cultures overnight.
2. Inoculate 10 ml of prewarmed medium (including antibiotics) with 500 ml of the overnight cultures, and grow at 37℃ for 30 min, with vigorous shaking, until the OD600 is 0.5-0.7.
The short second growth from an aliquot of the saturated culture ensures that all cultures are grown to a similar density before induction.
Larger cultures can be scaled up accordingly. If expression levels are very low (<1 mg/liter), it may be necessary to purify the protein from up to 50 ml culture.
3. Induce expression by adding IPTG to a final concentration of 1 mM.
Do not add IPTG to the culture which will serve as a noninduced control. If a time course of expression is being taken, the t=0 sample serves as the noninduced control.
4. Grow the cultures for an additional 4–5 h, and transfer to microcentrifuge tubes. Harvest the cells by centrifugation for 1 min at 15000 × g, and discard supernatants.
If a time course of expression is being performed, take 2-ml samples at hourly intervals, collect the cell pellets, and store at - 20℃ until all the samples are ready for processing.
5. Resuspend cells in 400ml buffer B. Lyse cells by gently vortexing, taking care to avoid frothing.
The solution should become translucent when lysis is complete.
The culture volume used depends on the expected expression level. When the protein is expressed at very high levels, (50–100 mg/liter) a 5´-concentrated cell lysate (resuspend the pellet from a 2-ml culture in 400 ml buffer B) can be used. 400 ml of a 5´-concentrated cell lysate in buffer B will contain approximately 100–200 mg of 6´His-tagged protein. For lower expression levels (1–5 mg/liter), 10 ml of cell culture should be used for a 25´-concentrated cell lysate. Resuspend the pellet from a 10-ml culture in 0.4 ml buffer B (0.4 ml cell lysate = 10 – 50 mg) of 6´His-tagged protein.
6. Centrifuge the lysate for 20–30 min at 15 000 ´ g to remove cellular debris, and transfer the supernatant to a fresh tube.
7. Equilibrate a Ni-NTA spin column with 600ml buffer B. Centrifuge for 2 min at 2000 rpm (approximately 700 ´g).
8. Load the cleared lysate supernatant containing the 6´His-tagged protein onto an equilibrated Ni-NTA spin column
9. Centrifuge the Ni-NTA spin column for 2 min at 2000 rpm (approx. 700 ´ g), and collect the flow through.
It is important not to exceed 2000 rpm (approx. 700 ´ g) when centrifuging Ni-NTA Spin Columns. Higher speeds reduce binding time, resulting in inefficient binding.
Save the flow through for SDS-PAGE analysis.
10. Wash the Ni-NTA spin column twice with 600 ml buffer C. Centrifuge for 2 min at 2000 rpm (approx. 700 ´ g)
Save the flow-through (wash fractions) for analysis by SDS-PAGE to check the stringency of the wash conditions.
It may not be necessary to repeat the buffer C wash. The number of wash steps required to obtain highly pure protein is determined primarily by the expression level of the 6´His-tagged protein. When the expression level is high, two wash steps are usually sufficient for removal of contaminants. For very low expression levels or highly concentrated lysates, three wash steps or washes with buffer D may be required to achieve high purity.
11. Elute the protein with 2 ´ 200 ml buffer E. Centrifuge for 2 min at 2000 rpm (approximately 700 ´ g), and collect the eluates.
Most of the 6´His-tagged proteins (>80%) should elute in the 200 ml eluate, especially when proteins smaller than 30 kDa are purified. The remainder will elute in the second 200 ml. If the protein should be more concentrated or if the expected expression is low, elute in 100-150 ml aliquots and/or do not combine eluates.
12. Add 2.5 ml of 5´ SDS-PAGE sample buffer to 10 ml aliquots of all samples, including the undbound fractions, and boil for 5 min at 95℃.
13. Analyze the samples by SDS-PAGE.
