From your 5X stock prepare 1X Equilibration Buffer (50 mM sodium phosphate pH 7.0, 300 mM NaCl)—you will need about 1 L total for resuspending the cells and washing the microfluidizer between samples.
Place the pellets in the tubes on ice and resuspend each completely (no chunks) in 10 mL of 1X Talon Equilibration buffer minus imidazole. Resuspend one pellet of the wild-type and one each of two ncAA-mutant proteins (week 3). Take your resuspended cells on ice, your bottle of 1X Equilibration Buffer, and Oakridge centrifuge tubes to the Core Lab in ALS 2124. Your TA will show you how to use the microfluidizer. We will send groups up one at a time. To finish lab on time, you must microfluidize in the first hour of lab. Groups will not be allowed to begin microfluidizing after the first hour. You also need to get all the way through to eluting the protein from the resin before lab ends. Work efficiently!
Once you have recovered your lysed cells, return to the BB Teaching Lab and centrifuge your samples in the Sorvall SS34 rotor for 18,000 rpm for 20 minutes, 4ºC. Be sure to balance the tubes first.
Preparation of TALON Resin and Sample Binding:
While microfluidized cells are centrifuging, prepare and wash the BD TALON metal affinity resin, following the protocol outlined in the Talon manual (specifically, the protocol for a wild-type purification at pH 7.0). This manual also includes the recommended amount of bed volume of resin to use—anticipate that there will be at least 20 mg of protein per 100 mL of culture for wild-type protein (the yield varies depending on how stable each protein is and the efficiency of translational machinery so each TAG site will be different depending on its location and the ncAA incorporated). Once the resin has been washed and combined with the sample in a 50 mL Falcon tube, bind the sample to the resin for at least 30 minutes (at room temperature or colder) with rocking. Once the sample has been bound, it can either be washed in batch or be applied to the column and washed extensively (2 x 10 mL approximately). The extent of washing can be monitored with Bradford Coomassie stain—if a blue color appears when 500 mL of stain is combined with 20 mL of eluent, then there is likely still non-polyhistidine tagged protein still being washed from the resin. Once no blue color appears, elute the purified protein with 1.5-2 mL of elution buffer. This is a convenient volume to use for the next step, desalting.
- ADVICE: Adding the 1.5- 2 mL of elution buffer in smaller portions to the resin tends to improve yield of purified protein.
Store the eluted protein fractions at 4˚C (not frozen!!!) or on ice until you are ready to proceed with the next step.
To desalt the protein sample into the storage buffer, follow the procedure outlined in the PD-10 desalting columns manual. Anticipate that it will take approximately 20 minutes to wash the desalting column with the storage buffer. You may do this during the next lab session.
Sonicating Method to Lyse Cells (alternative method):
Place the pellets on ice and add an appropriate amount of wash buffer. Combine all pellets of the same protein type (i.e., wild-type vs. the two ncAA-mutant pellets) into a single 50 mL conical tube.
- ADVICE: Since the resuspended cells will eventually be centrifuged in special high- speed tubes that hold 40 mL, make sure that the total volume used to resuspend all pellets of a protein type does not exceed 40 mL, so that all pellets of a single type can be in the same tube. Use 15 mL lysis buffer/25 mL cell pellet
Once the pellets have been combined, add ~20 mg of lysozyme (not exceeding a final lysozyme concentration of 1 mg/mL) to the resuspended cells. Mix the lysozyme into solution by gentle inversion only—do not shake, foam (i.e., whip air into), or warm the samples, as the protein may be unstable when exposed to air or heat. Any remaining pellet pieces can be resuspended with brief sonication.
To lyse the cells more completely, they can be sonicated. Sonication ruptures the cell walls much the same way that standing next to a concert speaker can rupture eardrums. Therefore, ear protection should be worn throughout this process. Use the highest power setting to sonicate without foaming the sample. Moving the sonicator tip near the surface of the solution will make it foam, and touching the sonicator tip to the plastic tube will prevent it from lysing the cells well. Do not sonicate in glass. Sonicating each sample for three one-minute intervals should be sufficient. Do not let the sonicator warm the sample to above room temperature—if the sample starts to warm, stop sonicating until it cools down.
To remove cell debris, the samples must be centrifuged really fast in special high-speed tubes with caps. After ensuring that the tubes are balanced in the rotor (check the tubes with a balance) and that they are not cracked or structurally compromised, spin them as fast as the centrifuge will allow for 30 minutes.