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…