Purification of 6X His Tagged Proteins

Purification of 6XHIS proteins:

 

1. Spin cells harboring 6XHIS proteins in large bottles in Beckman Centrifuge 6000rpm 15 min.

2. Dump supernatant

3. Resuspend in 3ml Cell extraction buffer.

4. Grind protein in mortar and pestle plus liquid Nitrogen. (at least 10 min.) until becomes a fine powder.

5. Transfer frozen powder to 15ml conical tube and bring volume up to 10 ml.

6. Transfer approx 1ml to microcentrifuge tube and spin for 15 min at full speed 4C.

7. While spinning pre-equilibrate Ni-NTA resin with extraction buffer by washing 3X in extraction buffer and resuspend in 50% slurry.

8.  Pool all the supernatants from step 6 into a 15ml conical (save 100ul of supernatant =Load) and add about 300 ul of  Ni-NTA resin to the supernatant. Save some of the pellet (= insoluble fraction).

9. Bind 6XHIS protein to resin for 20min to 1 hour room temp.  Spin and save 100ul of the supernatant= unbound fraction.

10. Wash resin 5 times with extraction buffer (5ml each time).

11. Elute with low pH elution buffer (1ml each elution).  Elute 5 X.

 

 

Extraction buffer: 10mM imidazole, 500mM NaCl, 50mM NaH2PO4, pH8.0, (Optional components:  0.5mM TCEP, 1x protease inhibitor cocktail -Complete PI EDTA free tablets; Benzonase Nuclease HC,3 µl per 60ml extraction buffer).

 

HIS tagged proteins can also be extracted with Tris buffers.

 

Preparing Protein Samples:

 

1. 50ul 2X sample buffer  +  50 ul Load

2. 400ul 2X sample buffer +  pellet of insoluble fraction

3. 50ul 2X sample buffer + 50 ul unbound fraction

4. 50 ul 2X sample buffer + 50ul of each elution (1-5).

 

Boil samples at 10 min. with lid locks.

 

Load 20 ul of each on 10% SDS gel

 

1. Marker (only 10ul)

2. load

3. insoluble

4. unbound

5. elution 1

6. elution 2

7. elution 3

8. elution 4

9. elution 5

10. blank—load 20ul sample buffer

 

Run gel 100volts  for 2hours 15min or until the dye just reaches the end of the gel.

Stain gel with 100ml Coomassie Blue Stain (at least 1 hour). De-stain overnight with multiple changes.