One Day Purification

Goal

To lyse cells and then extract in order to purify the proteins of interest using a nickel (Ni2+) protein affinity column.

Before you start

1. Make sure the lysis buffer, wash buffer, and elution buffer are made and enough for experiment (Premade by us or Nicole, found in MEE 4ºC fridge)

   

2. Ask Eric to print appropriate labels beforehand:

   1.5 mL microcentrifuge tubes labels: Lysis 1, Lysis 2, Wash 1, Wash 2, and Fraction 1 to 8 (8 different labels)

What You Will Need:

LOCATION: MEE LAB

• -80ºC frozen induced E. coli cell pellets
• One 15 mL Falcon tube
• Six 50 mL Falcon tubes (to contain reagents in ice bucket)
• Timing device (like a phone)
• Tubes labeled as follows (you’ll need a set of all these tubes for every protein you’re purifying. e.g If you have 2 proteins then double the number of tubes)

  ‣2 x 50 mL conical tubes - Label: Lysis Buffer Flow-through and Wash Buffer Flow-through 
  ‣2 x 50 mL conical tubes - Label "Resin Beads"
  ‣4 x 1.5 mL microcentrifuge tubes. Label: Lysis 1, Lysis 2, Wash 1, Wash 2
  ‣8 x 1.5 mL microcentrifuge tubes. Label 1 to 8 (These will be the 8 fractions)

• Serological pipettor with the following tips

  ‣4 x 25 ml
  ‣2 x 10 ml 
  ‣4 x 5 ml   

• Large ice bucket filled up to 75%. Put the following things in bucket:

  ‣Proteins to be purified
  ‣Nickel agarose high-density resin beads (4ºC MEE fridge; keep cold at all times)
  ‣2 X 50 mL Lysis buffer in 50 mL conical tube
  ‣50 mL Wash buffer in 50 mL conical tube
  ‣15 mL Wash buffer in 15 mL conical tube
  ‣30 mL Elution buffer in 50 mL conical tube
  ‣Plastic protein column and twist-stopper for each protein

Procedure

1. Move these tubes, pipettor, and ice bucket to Bio Superlab

   LOCATION: BIO SUPERLAB

2. Have Eric turn on large ultra-centrifuge centrifuge and set it to 4ºC. (It needs to reach this temp before you use it later on)
3. Add 20-30 mL lysis buffer to each cell pellet for the protein samples you have
4. Resuspend the cells via vortex until the mixture is smooth to avoid clumps of the pellet. The mixture must be completely smooth.May take up to 10 minutes but is Very important!
5. Find the warning signs that indicate that the SONICATOR IS ON
   • this should be on the benchtop on the right of the door in the nanodrop room
6. Tape this warning sign to the outside doors of Nanodrop room, so that others will not unexpectedly enter the lab from the hallway.

   Important Safety Measure

   • Sonicators are high-frequency sound generators used to disrupt cells or shear nucleic acids. Laboratory personnel must be concerned about two of the major hazards associated with sonicators. The first hazard is hearing damage caused by high frequency sound (waves in the 20,000 Hz range). The second is the generation of aerosols from the sonication process. Actions you can take to reduce hearing damage is to wear earphone-type sound mufflers to protect your hearing while sonicating. *** Do not sonicate in a room where people are not wearing ear protection and wear earphone-type sound mufflers all the time while sonicating.
7. Put on ear protection!
8. Set up a beaker of ice that then holds your cell/lysis buffer mixture. Sonication generates heat; we want this mixture to remain at 4ºC at all times.
9. Clean and dry sonicator probe using ethanol and Kimwipes before use.
10. Fully immerse the sonicator tip in tube with cell mixture, but make sure the tip does not touch any tube walls.
11. Sonicator operating settings:
    • Put sonicator at a power level of 7
    • Press the ON button on the sonicator and to start it, flip switch on the front of device from REMOTE to CONTINUOUS
    • Operate 30 seconds on, 30 seconds off. Do for 10 minutes total ( 5 min sonication overall)
    • If you have more than one sample, it is important to clean the probe with ethanol when switching between them.
12. Spin down the sonicated cells on the chilled ultra-centrifuge (it should be 4ºC at this point)
    1. Use small, cylindrical centrifuge tubes

       • These will be located under the swinging bucket centrifuge near all the nitrile glove boxes.
       • Make sure the tubes are identical (same brand and type)
       • Before using tubes, place them in the centrifuge to make sure that they can fit in, because not tubes will fit.

    2. Label centrifuge by writing on a piece of colored tape and sticking it on
    3. Ultracentrifuge is very sensitive, so all weights must be properly balanced:

       a) Put an empty beaker on the scale (this will help keep the tubes upright)

       b) Tare scale so that it reads 0.000 g

       c) Weigh out the empty centrifuge tube (should include cap and label in weight)

       d) Add all the contents from the tube with sonicated cell mixture to centrifuge tube

       e) Record total weight

       f) Weigh other empty centrifuge tubes to match the recorded weight.

       • Balance tubes can be filled with water.
       • For other sample tubes, if they differ in weight with the first measurement by ≈ one gram, add 1 mL of lysis buffer in the lower weight tube and measure it again. Tubes should be within 0.1 grams to each other. Measure the filled tube with cap and label.

    4. Ultracentrifuge settings: 14,000 RPM for 45 minutes; have Eric double-check your tube weights and start the ultracentrifuge.
13. While sonicated sample spin, start next steps

    
    Nickel Column Equilibration

14. Get Nickel agarose high-density beads ice bucket and shake well! These beads will settle out very, very quickly.
15. Equilibrating column with lysis buffer (the following is needed for each protein sample):

    • The nickel bead solution is termed slurry, which is a combination of resin beads and ethanol. 1 mL of resin beads == 15 mg protein (good for about 2 L of induced, grown E. coli). We use 2 mL of resin beads to get as much protein out as possible; as the resin is in 50% ethanol, we need 4mL of resin.

    1. Put 4 mL of slurry in a 50 mL conical tube. Shake the nickel bead slurry right before pipetting.

       NOTE: Do not leave any of the reagents out of ice bucket

    2. Use spinning bucket centrifuge (next to nitrile glove boxes) at 3000 RPM for 2 minutes (Be sure to balance centrifuge properly using methods described in STEP 12.3)
    3. Carefully remove ethanol supernatant with a P1000 pipette (Be careful not to disturb the pellet of beads on the bottom. This is tricky!)
       • Try cutting of a small piece of the P1000 tip to make the opening wider to reduce suck-up rate
    4. Discard ethanol in the sink
    5. Add 2 mL lysis buffer
    6. Mix and spin down at 3000 RPM for 2 minutes
    7. Carry out STEPS 3-6 a total of 2 more times, then proceed to next step for the final spin.
    8. For final spin down, carry out STEPS 3-4. Do NOT add any lysis buffer because you need the resin to be concentrated as much as possible.

    
    Combining Column with Lysate

16. Gently pour cell lysate from the sonicated cell mixture centrifuged in STEP 12 into 50 mL tube that has the equilibrated resin beads.
17. Leave at least 1 mL of the cell lysate so that cell debris does not mix into with the lysate. Very important step
18. Pipette up and down to resuspend the resin beads in the cell lysate
19. Seal with parafilm (found in the drawers under the sink near the door)
20. Place flat on orbital shaker for 30 minutes (Good stopping point for lunch)

    
    Disposable Column: Lysis Stage

21. Put the following tubes on appropriate racks to be take to the cold room:
    • The four 1.5 mL microcentrifuge tubes labeled Lysis 1, Lysis 2, Wash 1, Wash 2
    • The two 50 mL conical tubes labeled Lysis Buffer Flow-through and Wash Buffer Flow-through
    • The eight 1.5 mL microcentrifuge tubes labeled 1 to 8 (These will be the 8 fractions)

    LOCATION: BIO SUPERLAB COLD ROOM
    NOTE: Always keep the door closed!

22. Get the disposable column and carefully twist off the end. Get the lid ready. Attach the separate twist-stopper. Ensure that the stopper is closed, with the twist part perpendicular to the column.
    • Watch optional short video
23. Set up nickel column with clamps holding it in place (see picture below)

    

24. Pour in resin and proteins along the side of the column. Rotate tube to get resin out
    NOTE: Avoid trapping air bubbles.
    NOTE: Do NOT let the resin get dry, keep it hydrated at all times
25. Open bottom and let all liquid pour through into 50 ml tube Lysis Buffer Flow-through
26. Take small volume (about 500 uL) of this flow-through and put in 1.5 mL microcentrifuge tube labeled Lysis 1,
27. Take Lysis 1 to Nanodrop to obtain absorbance readings
    1. Blank with lysis buffer and measure it (to assure Nanodrop is clean and that there is no preexisting absorbance)
    2. Select "Overlay spectra" between readouts.
    3. Record the value for 10mm absorbance at 260 nm wavelength
    • The aim is to see a strong absorbance at 260 nm due to oligonucleotides
28. Go back to the cold room and add 10-20 mL lysis buffer to the column
29. Collect the last few drops from the column in the 1.5 mL microcentrifuge tube labeled Lysis 2
    • The aim is to see the strong absorbance at 260 nm completely disappear, implying oligonucleotides are removed
    • If absorbance at 260 nm is higher than zero, then keep adding lysis buffer and measuring the absorbance of sample until it does (it should not more than 10-20 volumes, roughly 10-20 mL)
      NOTE: when adding buffer directly to column, use a serological pipette to pour buffer down the walls of the column to avoid disrupting the resin beads.

    
    Disposable Column: Washing Stage

30. Replace the 50 ml tube Lysis Buffer Flow-through from under the column with the 50 ml tube labeled Wash Buffer Flow-through
31. Add 20-30 ml of wash buffer to the column and let it all run through
32. Take small volume (500 uL) of this flow-through and put in 1.5 mL microcentrifuge tube labeled Wash 1
33. Take Wash 1 to Nanodrop to obtain absorbance readings
    1. Reopen the Nanodrop program
    2. When prompted about which initial setting to use, click “A280 Protein” setting to acquire the readings
    3. Select "Overlay spectra" between readouts.
    4. Blank with wash buffer and measure it
    5. Record the value for 10mm absorbance at 280 nm wavelength

    • The aim is to see some initial 280 nm absorbance due to unwanted proteins:

34. Go back to the cold room and add 10-20 mL wash buffer to the column
35. Collect the last few drops from the column in the 1.5 mL microcentrifuge tube labeled Wash 2
    • The aim is to see the initial absorbance at 280 nm completely disappear, implying unwanted proteins are removed
    • If absorbance at 280 nm is higher than 0.5, then keep adding wash buffer and measuring the absorbance until it does (it should not take more than 10-20 volumes, roughly 10-20 mL)

    
    Disposable Column: Elution Stage

36. After washing step is complete, cap the 50 ml tube Wash Buffer Flow-through and get the 1.5 mL microcentrifuge tubes labeled 1 to 8 ready to collect the fractions
37. Add 15 mL elution buffer to the column and prepare to collect the flow-through in each of the fraction tubes
38. Collect 8 different fractions, each at 1 mL, and close the column, leaving about 7 mL remaining in the column
39. Take the fractions to the Nanodrop and measure absorbance of each of 8 fractions
    1. Reopen the Nanodrop program
    2. Use “A280 Protein” setting to acquire the readings
    3. Select "Overlay spectra" between readouts.
    4. Blank with elution buffer and measure it
    5. Measure all 8 and watch for the trend of a bell curve

       • If the graph does not taper off back to baseline, go back to cold room and collect the last 7 mL in the column left in STEP 34 until readings go back to baseline

    
    Deciding Which Fractions to Keep

40. Look at the Nanodrop graph, pool fractions where 280 nm absorbance readings are highest
41. Pool (put in one tube) high fractions together into 15 mL conical
    NOTE: Usually the top 2-3 fractions are pooled together
42. Repeat Nanodrop measurement of this pool