Gel Filtration on Michael’s Domain

Increased resilience

Tue, 27 Nov 2018 00:00:00 +0000

I strongly believe that companies are genuinely interested in getting critical feedback. At the very least, it should be part of their corporate survival instinct. When dissatisfied customers simply switch to an alternative vendor without giving feedback, the damage is already done. That’s why I always give feedback if a product does not meet my high-quality expectations.However, this time I am writing about a product I have been very satisfied with, namely the new GE Healthcare Superdex 75 Increase 10/300 GL . It is a gel filtration column and an iteration of the previous Superdex 75 10/300 GL , which we have been using for the last 30 years. The biggest advantage of the “Increase” is the higher pressure-resistance. A few weeks back an overworked grad student forgot to close the column after use and returned it to the fridge, where the 20% ethanol slowly evaporated over the following 2 weeks. The fridge is ventilated and when I needed the column, the damage was already done: There was a perhaps 8 mm gap and a dry zone of approximately once inch had been developing. I immediately filled up the dead volumes with degassed 20% ethanol and started a very slow run (0.05 ml/min) for several hours, after which is switched to degassed water and then to buffer. After letting it run for about 2 days the gap was reduced to about 3 millimeters. The remaining gap was removed by adjusting with the top adapter (I needed to screw it down as much as possible).Now I needed to test the “repaired” column. I did both a functional test (separating two proteins in PBS) and the acetone test (injecting 100 µl 2% acetone in water). I was massively surprised when the aceton test showed about 19000 theoretical plates (which is more than we ever got with our old Superdex 75 columns). And the separation of the two proteins (RNaseA and BSA) showed that the column is still fully functional. However, we cannot tolerate any gel compression, since the ability of the adapter to correct for it is maxed out (GE Healthcare used to produce a longer adapter, which would allow us to compensate even further, but they chose to discontinue this product).Both the grad student and myself were relieved after getting these results because buying a new column would have set us back by 2200€ and since we have no dedicated funding to operate our core facility , I would have been forced to offload this expense to the grad student’s laboratory. Unfortunately, I did not take a picture of the damaged column as my instinctive reaction was to immediately start the rescue. However, the column in the picture below is the damaged column after the rescue operation was complete.

Purification and Characterization of Recombinant Proteins

Mon, 20 Mar 2017 00:00:00 +0000

We are organizing (again) a practical hands-on protein purification course from December 4th to 20th. We maximally can accommodate 16 participants, which will form groups of 2 to 4 participants. Each group needs 3 full days to go thru the practical exercises, but day 3 of the course will be overlapping with day 1 of the next group. Venue is Biomedicum Helsinki, rooms A516a1 (where the machinery is) and B318a/b (our lab). We will purify a protein (VEGF receptor 3) using a two-step protocol (affinity chromatography + gel filtration) on the the Äkta Avant FPLC device. On the third course day we’ll assay its interaction with its ligand (VEGF-C) on the ITC (isothermal calorimetry) device. We have given a similar course in 2015 ( http://www.helisci.fi/hbgs/FPLC2015/) . This new course ( https://courses.helsinki.fi/en/DPBM-135/120171139 ) will have a similar structure, but we expand it by one day to analyze protein interactions making use of the new isothermal calorimetry device of our B3P core facility. The documentation and results will be also available from here . Since we can run maximally two samples at a time (we have “only” two FPLC machines), we will have to split the participants into groups (of 2-4 students/group) and repeat the 3-day course several times depending on the number of participants.

HiLoad 26/60 Superdex pg gel filtration chromatography column performance

Mon, 11 Apr 2016 00:00:00 +0000

The most common protein purification technique that we use is gel filtration (also called size exclusion chromatography). In gel filtration, proteins are separated by their size (or more correctly by their “Stokes radius”, which is largely determined by their size and shape). For gel filtration of large protein amounts, we bought GE Healthcare’s HiLoad 26/60 Superdex 200 prep grade about 9 years ago (the 26/60 has been replaced by HiLoad 26/600, but both are almost identical). With this column you can very cleanly separate two proteins that have a size difference of 100%. Therefore it is suitable to separate monomeric from dimeric versions of the same protein. The columns “expiry date” was 2012-03. Our experience is that under proper handling, such a column can easily reach a life span of 10 to 15 years (we don’t use it very often, we store it in 20% ethanol, if we don’t need it for longer periods of time, we keep it at +4°C).

FPLC Protein purification course

Mon, 04 Jan 2016 00:00:00 +0000

Eight postgraduate students registered for the FPLC protein purification course , which took place in December. If I learned anything, than that protein purification during a course should be done ALWAYS with a protein and a protocol, that has been used before successfully MANY times. Student-provided proteins are a great source for learning, but the time restraints of a course format did not allow us to finish the purification of these proteins during the course.Technical Problems with the new Äkta Avant 25In addition, the Äkta Avant 25 , that we newly purchased from GE Healthcare this summer, broke TWICE during the course. First the controlling computer broke (RAID failure). HP delivered the replacement drive within 24 hours and after the RAID had rebuilt itself, we could continue the course. However, during the first run after this incident, the Äkta ran into an overpressure problem. We identified a faulty flow restrictor as the cause, but we did not want to continue as we have had severe problems with air bubbles in previous runs. Therefore we performed the runs on the old Äkta Explorer 100 . GE Healthcare quickly had their service engineer check out the system, but since he did not have a spare with him, we had to wait until Monday 14.12. until the Äkta Avant was again fully functional.What we purified: soluble VEGFR-3 (VEGFR-3/Fc) and HepsinWe did purify soluble human VEGF receptor-3 (the first three domains of its extracellular domain connected to the constant Fc part of human IgG). This is a purification that we have done many times. It is equivalent to the purification of antibodies using Protein A sepharose.We had prepared in advance conditioned cell culture medium. We produce most of our proteins in insect cells (mostly Drosophila S2) and the VEGFR-3/Fc had been secreted by the S2 cells into the medium after induction of the metallothionein promoter with 1 mM Cu2+ for about 4.5 days. The preparation of the medium for purification consists only of 1) getting rid of the cells by centrifugation and 2) filtration to remove precipitates and other small particles that might clog the column. There is no need to adjust the pH.Rapid neutralization after low pH elution IS IMPORTANTWe ran the medium over a disposable 5-ml HiTrap recombinant Protein A column over night at about 1ml/min and eluted with a low pH buffer. The eluted 2-ml fractions were immediately neutralized with 400µl 1M Tris pH 8.5. Here we made a small mistake during one of the two purifications. GE Healthcare had not released the casettes for the 5-ml-collection tubes (they still have not done so even though they did promise them already for September 2015) and we used 15-ml Falcon tubes to collect 2-ml fractions, into which we had pre-aliqotted 400 µl of the neutralization solution. However, the mixing in these tubes was not efficient and the prolonged exposure to low pH resulted in a partial damage to our protein. This can be seen when comparing the size exclusion chromatograms of Group 2 versus Group 4: For Group 4 the first peak (aggregated protein) is much larger and more heterogenous compared to the same peak for Group 2.In fact, when VEGFR-3/Fc is eluted by low pH from protein A columns, it always precipitates at higher concentrations soon after elution, but dissolves again upon neutralization. This did not happen in the fractions 5.A.3 and 5.A.4 (Group 4) due to the inefficient mixing of elutate and neutralization buffer in the 15-ml-Falcon tube (the fraction size of 2 ml was probably to blame as well; 1 ml would have been better). During the run for Group 2, we removed the tubes immediately after the run had ended and thereby mixed the buffers, while for Group 4 the run finished during night time and the eluate remained largely unmixed until the morning.Alternatively, we could have eluted with a highly concentrated chaotropic salt at near-neutral pH (which we’ll do next time in case we have an automated run where the elution happens in the middle of the night). Pierce offers a (proprietary) “gentle” elution buffer of pH 6.6. I don’t know the Pierce buffer composiiton, but it is a highly concentrated solution of some chaotropic salt. 3M potassium/sodium thiocyanate or 4M magnesium chloride in buffered solutions around pH 7 are frequently used chaotropic salts for this purpose.HepsinThe student-provided proteins were challenging. First, their concentrations in the starting material was very low. While we can see clearly the protein when the VEGFR-3/Fc conditioned medium is run on a PAGE gel and stained with Coomassie, no such band is visible for the Hepsin. In addition, it appeared that a significant fraction of the protein seems not to contain the histag (anymore) and therefore is not captured with the first purification step. The fraction of Hepsin-H6 that does bind to the Ni2+ sepharose elutes already at an imidazole concentration of 20 mM, which makes washing the column challenging. The Hepsin with the longer histag (H10) survives the 20 mM imidazole wash, but it suffers also from low expression levels.Below are the chromatograms of the individual runs and the annoteded images of the Comassie-stained PAGE gels. The detailed protocol for the operation of the Äkta Avant 25 is still under preparation…