8 Comments

Protein purification: From cell pellet to freezer in less than 12 hour – Part 2

Ion exchange chromatography - Mark that peak!

Continuing the 12 hour purification…

Ion-exchange column – 5/50GL Mono Q

11:25 – Using AKTA purifier FPLC (GE healthcare) I’ve prepared and conditioned the high resolution Mono Q 5/50GL column with low salt buffer (50mM NaCl) and prepared our special inlet buffer valve position A8 (A18).

11:40 – Through the buffer valve A18 I’ve loaded the column with elution sample so I will have a good balance between time and peak resolution. Elution was conducted at 40%/40’, at 1ml/min, thus eluting the proteins at relative low volume. First run was performed with 5ml sample loading. Logged the run specs in my Web based knowledge management system (BioKM).

12:00 – Running SDS-PAGE of the First run main peaks and with the previous Ni-NTA column elutions (see figure). At 160V this gel run was expected to end within 75min. Logged the lanes order and specs in BioKM.

12:10 – Elution sample separation was continued while keeping the peak fractionation as much as possible similar to the first run.

Concentrating & freezing samples

13:30 – After doing quick staining I figured that the last fraction is the most clean, and started pooling the already fractionated samples (logged the fractions keepers in my BioKM account).

13:35 – Started concentrating the keeper fractions in 10,000 MWCO as is (no need to switch buffer as the conductivity measured fits the required ionic strength).

14:10 – Seminar. Brought my laptop with me and I wrote some great ideas relevant to my projects in my BioKM account.

15:05 – Continued pooling keeper fractions and concentrating them.

18:30 – Fihished ~7 runs and pooled the last keeper fractions. Measured Absorbance at 280nm and got 10mg/ml. On the dot, which is exactly the concentration I needed. Logged the value in BioKM.

18:35 – Finished preparing 80 eppendorf tubes with a predetermined short identification code and fetched the liquid nitrogen.

18:55 – Finished preparing aliquots & freezing my samples. Logged in BioKM: number of vials, the aliquot volume and protein concentration.

19:00 – Full sample box was placed at -80°C. Logged in BioKM the position of the protein box and Wrap things up.

19:15 – Locked the lab and going home singing…

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8 comments on “Protein purification: From cell pellet to freezer in less than 12 hour – Part 2

  1. Just out of curiosity, how long do your proteins last in -80? Indefinitely? The two proteins I work with cannot be frozen and both degrade in a matter of days, so I make them fresh every time I need them; the whole concept of preparing proteins ahead of time and freezing them is totally foreign to me!

    • From my own experience I’ve not yet experienced a problem with protein samples in the -80c and I have samples that are stored almost 2 yrs.

      “The two proteins I work with cannot be frozen” – I am curious, if you do not mind tell me:
      1. How do you freeze them?
      2. What happens to them once frozen?

      Chen

  2. We’ve been trying since the protein was first purified in our lab in 2002 with no success; for one protein, regardless of the conditions we have tried, the protein immediately precipitates, likely due to its degree of disorder (we are also mostly unable to dialyze out the imidazole due to the same reason). The other protein, a helicase, loses enzymatic activity with freezing.

  3. We have tried freezing in liquid nitrogen, in dry ice/ethanol baths, straight into the -80, etc. We have tried a variety of thawing techniques with the helicase but still lose activity; the disordered protein I study precipitates at any point above 30c, so we have never tried incubating it after freezing.

    • I would try to incubate it at 27c (disordered protein) and see if it survives. I guess the helicase have homologue proteins which were characterized – have they detailed how they purified the protein?

  4. The only other purification scheme for a similar protein uses a column which is no longer produced! We have no problems with the purification step, just the storage… so for now, we are stuck purifying our proteins on a weekly basis. Fortunately, we have most of this automated from column to column, so there is very little hands-on once we load the protein onto the first column.

    • Indeed, automation is the reasonable method of opertation in such cases. Sometimes authors discuss specific problems they encounter with a certain protein as it reveals the protein’s biochemical or functional characteristics.
      Wonder how it behaves in 50% glycerol at -20c?

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