Protein Purification on Michael’s Domain

Producing proteins better than the pros

Thu, 31 Oct 2024 00:00:00 +0000

Congratulations, Laura & Akseli! Your article in Scientific Reports ( https://doi.org/10.1038/s41598-024-77104-3 ) is a solid piece of work that has not only relevance for cardiovascular diseases but also for the work that we are doing on lipedema/lipoedema (and therefore, potentially even for lymphedema, which has been the main focus of our research in the past). There are astonishing similarities between coronary artery disease and lipedema, because both can be regarded as lipid storage disorders.And thanks to Khushbu and Betül for helping Laura produce and purify such a high-quality LCAT protein that worked better than the protein preps that are commercially available! Perhaps much of the secret was the high expression levels that we could reach compared to other systems. For the first time, we tried out HEK293T cells with a custom CHO vector ( pCHOKE-B-LCAT-H6 ), and it worked surprisingly well. pCHOKE-B was assembled from scratch and we incorporated several advancements made over the years compared to regular CHO expression vectors. The vector contains

Bio-Rad fixes our NGC

Wed, 20 Sep 2023 00:00:00 +0000

UPDATE (situation Dec. 21st, 2024)

We just survived our protein purification course (DPDR-305, see also my other blog posts related to teaching . After having done about 25 runs with the “repaired” Bio-Rad NGC, I sadly have to conclude that the most significant issue remains: that the FPLC becomes unresponsive to commands issued manually. Also, the randomness of these disconnects persisted. Only one student group had problems, but we had several incidents during that single day. That, sadly, concludes our short stint into Bio-Rad territory for protein purification. All of this indicates that any further investments into this device will be wasted time and money. We have neither too much time nor too much money. If Bio-Rad wants to do anything from their own initiative, I am happy to let them do whatever it takes to get the machine into a usable state, but we won’t actively pursue any further actions.The best way to keep your FPLC device in good shape is to have a maintenance contract. Although we have been able to get money from our university to buy a top-of-the-line FPLC twice in the last 30 years, getting money for a service contract is much more difficult. One of the many reasons is that most grant periods are shorter than service contracts, which only make sense if you make them over several years.

Showdown: GE Healthcare's Äkta versus Bio-Rad's NGC

Tue, 07 Feb 2023 00:00:00 +0000

I have been purifying proteins since 1996. I worked on an Äkta Explorer until 2015, when we upgraded to the Äkta Avant . In 2020, my lab moved, and we inherited a Bio-Rad NGC , which we have been using now for 2 years.We encountered many problems with the Bio-Rad NGC. At first, I thought that this might be normal when switching systems. I expected the problems to disappear one by one. After all, we also had problems when we switched from the Explorer to the Avant.However, even after two years and dozens of purification runs, the problems with the NGC don’t seem to stop. Whenever ẃe solve a problem, a new, previously unknown problem appears. And differently to GE Healthcare, Bio-Rad’s customer service is not even close to what we have experienced with GE Healthcare. When our IT could not connect the Äkta to our university’s network, GE Healthcare sent an engineer from their Munich crew to Helsinki to fix the problem. Appreciating the learning curve, GE Healthcare also offered free participation in one of their courses for somebody from our team. And their support was not limited to the warranty period! They really wanted us to be happy with their device. We don’t experience the same amount of support from Bio-Rad. It always feels like we have to coerce them into solving the problems we have with the NGC, and their response time is well below any customer expectations.This is the first of several blog posts about Äkta versus NGC. I hope will find the time to write in more detail about all our issues over the next few months.We decided in 2015 that we finally needed a new FPLC. Our Äkta Explorer was reaching end-of-life, and we had received about 100k funding to renew the FPLC of our core facility . The device was expensive enough that we needed to go through the official tendering process (at the time, the limit was 30,000 €, but it has been increased to 60,000 € by now). There were only two contenders: The Bio-Rad Discover NGC and the GE Healthcare Äkta Avant. The Bio-Rad was cheaper (82,810 € versus 95,470 €), but we decided to purchase the GE Healthcare device. One important reason was that all our users had been using the Äkta Explorer and its Unicorn software. Switching would simply be disruptive and require lots of support and time from our side. However, there were also technical reasons that made us prefer the Äkta over the NGC:Äkta Avant’s advantages

Updating GE's Äkta Avant to Windows 10

Tue, 18 Feb 2020 00:00:00 +0000

The Avant is the flagship of GE Healthcare’s Äkta line of FPLC (fast protein liquid chromatography) devices. We use it on a regular basis and it is available to everybody from the University of Helsinki.Our University decided to play nice with Microsoft and to disallow all network traffic for Windows 7 computers. Because our Äkta (bought in 2015) is in use by many different researchers, we rely on Microsoft’s Active Directory to authenticate users and to track the device usage. When the University really did shut down Windows 7 traffic in late January, we finally updated the HP computer from Windows 7 to Windows 10 . Before we did that we contacted GE in order to be sure to have a working FPLC. Although the build number of Win10, that our University is using (Windows 10 Enterprise version 1809, build 17763.1039) was not supported by any of the Unicorn 7 versions that GE has released over the years (see this software compatibility chart, which I cannot find anymore from the GE website: https://drive.google.com/open?id=1cA33lEqfEr0MIaA8r9TyoQFcxiHWuZsp) , we were assured that the software would continue working. Unfortunately, this was not true.**In-place upgrade or clean install?**We performed an upgrade of Win7 to Win10 in place. I never liked these. Even on slightly superior operating systems like macOS and Linux , the in-place upgrades have not always been smooth. A fresh start has always been the safer (and also faster) bet.Connection problemsAfter the upgrade, Unicorn 7 started to lose the connection to the Äkta Avant during ongoing protein purification runs. We clearly could see during manual runs, that specific commands would trigger a disconnect. The Äkta LCD display would black out and the device would reboot. Naturally, we suspected that this was a consequence of the upgrade to Windows 10. Because Unicorn 7.0.2 had not been tested to work with our version of Windows 10, we first wanted to try to upgrade to a Unicorn 7 version that had been tested with our Win10 build. I had already asked in December for that update from GE, but my request had been ignored and I had many other things to do at the time and never bothered to come back to the issue (in hindsight a big mistake).Software updatesSomething that Chrome and Firefox do weekly silently in the background (without the user noticing) takes in the case of GE almost 2 weeks full-time engagement by the user. A simple download might work for Bio-Rad , but GE requires a complicated, obfuscated, non-transparent, user-unfriendly, constantly changing, GDPR-non-compliant syastem, which is in addition hosted on servers that feel so slow that you constantly wonder whether your browser tab has frozen. What it’s worth, GE’s web pages have always been like this despite multiple redesigns and GE’s digital transformation. The email thread between me and various GE support addresses comprises as of today 60 emails (starting with my first support request on December 2nd, 2019). On top of this, there are also quite a few phone calls (since email seems to be often regarded as “non-urgent” by default).Privacy concerns about GE’s handling of customer dataAfter placing an order for the download, receiving a quotation, confirming the order and receiving an order confirmation, I finally received the “permission” to download the newest version of Unicorn (which is 7.5). I logged into GE’s “eDeliveryPortal” only to realize that I could see the software entitlements and downloads for many other GE customers, but none of my own. After some back-and-forth the 7.5 version finally showed up under my downloads. My license is still missing as of today. But why bother? I could use any of the other licenses (after faking the MAC address of my computer as the software apparently is locked to specific computer via the computer’s network card’s MAC address). This is clearly a violation of the GDPR as there is absolutely no reason why my data (entitlement ids, MAC addresses, Names, e-mail addresses) should be available to other customers. It might be that GE considers the University of Helsinki as a single customer; that would be a violation of the GDPR by design.**Searching for the system configuration files (i.e. firmware)**However, we had not updated the system configuration files, because I had not been able to find them from the GE LifeScience website . We were running version 3, while version 3.6 was the newest one. Notably, these files do also contain the firmware for the individual Äkta components. It appears that this download is nicely hidden. I needed help from GE in order to locate it. The GE life science website has a search function and the search even finds the download. However, it is perhaps the last item in a list of nearly 1000 hits (10 hits/page). One tip to GE: Google knows search! If you just would let Google index your complete web site, customers would find everything via a Google search. Many companies are using Google for their internal web site search. However, GE seems to expose only their product pages to search engine crawlers, but not their support pages.**Where are our licensing files?**After updating the instrument configuration, the problem persisted. After sending a system report (and then sending a second “extended” system report), GE concluded that most likely our in-place upgrade from Win7 to Win10 was to blame, because they could see some irregularities in the SystemEventLog.xml file. I had no reason to doubt this explanation and therefore requested a clean Windows 10 install from our IT guys. I then installed Unicorn 7.5 only to see that the problem had not disappeared. Just as a side note: We had a self-inflicted issue with our license file (.lic). The Unicorn installer software requests the installation of a license file, which I have never seen for download anywhere in the eDelivery portal (we have received these license files as attachments to e-mails directly from GE support staff, why are they not available for download as these are prone to be lost?). We should have made a copy of our license file before we erased our computer for the clean Win 10 install, but we missed that and I had to dig out the license file from a system backup, which I had luckily made in 2016 from the machine. And I am still searching for our license files for different system components (Classic Evaluation and Column Handling), which were not yet installed when I did the system backup.Down-grading to Windows 7In order to be sure that we did not deal with a hardware failure, we decided to downgrade the system back to Windows 7 and Unicorn 7.0.2. That was more difficult than expected, because we cannot anymore install Windows 7 on university machines. Luckily we had still one old, unused Dell desktop, that was runninig Windows 7. Unicorn 7 requires two network cards (one to communicate with the outside world and one for a separate 10. network to communicate with the Äkta Avant). We swapped the network interface card from our default computer to this old Win 7 machine. However, installing and running Unicorn requires access to the internet because the software is “phoning home”. Exactly that is disallowed for Win7 machines at our university. Hence I needed to set up a private network via my phone to allow Unicorn 7 to communicate with GE headquarters. Even worse, I needed to clone the MAC address of our default computer, because our Unicorn 7 software is apparently locked to a specific computer via the computer’s MAC address. And wireless internet access was a no-go, because the original MAC address was from a wired NIC. It is not possible to clone MAC addresses between cabled NICs and USB wireless adapters, because MAC addresses from wireless cards have a fixed prefix. Hence, I had a complicated setup from my phone via a computer to a (cable) router to the Win7 Dell computer.Windows 10 is not to blame - F-Secure is the culpritTo my surprise, the Äkta Avant kept crashing when Unicorn 7 under Win7 issued commands like “pause” or “end”. But then I realized that - unlike our old Äkta Explorer - the Avant uses regular NICs for the device communication and the University of Helsinki Win10 version is by default “enhanced” by remote control and security software. I went to the Control panel and uninstalled all software add-ons that had been installed by the university. Most notably F-Secure’s Client Security Premium was among the programs, that I uninstalled. After a reboot, I was unable to make the Äkta Avant crash. To confirm the finding, I popped the NIC back into our original default computer, rebooted and got stuck at the Windows BitLocker screen. Changing the hardware configuration inevitably triggers Windows to refuse booting. This did slow me down because it required me to engage our university’s IT department, which is chronically overworked and difficult to find. I selectively disabled the firewall from F-Secure Client Security Premium and the crashes instantly ceased also on Windows 10. It is very difficult to argue why our university would need F-Secure’s products. Windows itself comes (since XP SP2) with a very capable firewall and also has its own anti-virus software ( Windows Security Essentials / Windows Defender ), which is at least as good as its best competitors in addition to the fact, that it is free and fully integrated into Windows (and it does not require to stick third-party hooks deeply into the OS).Windows 10 is too slow on our 2015 hardwareThe only thing left is to upgrade our default computer to newer hardware. Our faculty had replaced older Win7 computers in 2019 with new machines, but this computer was 1 day too new to be included in the upgrade. However, it is already 5 years old and especially after the Windows 10 upgrade, it is very slow. When your protein elutes during your purification, you do not want fraction collection to start after a few seconds, but you want it to start instantly.

Protein Purification Course 2019

Wed, 31 Jul 2019 00:00:00 +0000

We are again hosting the DPBM protein purification course this December in our lab. Secure your place as this practical course is popular and there are only 16 seats. You can bring your own protein and we will individualize the course program based on your needs!More information: http://research.med.helsinki.fi/corefacilities/b3p/teaching.htmlRegistration : https://courses.helsinki.fi/en/dpbm-135/131042336

Practical Course: Purification and Characterization of Recombinant Proteins (DPBM-135)

Sun, 10 Dec 2017 00:00:00 +0000

Teaching material, results, etc. for the DPBM course “Purification and Characterization of Recombinant Proteins” ( https://courses.helsinki.fi/en/DPBM-135/120171139 ):

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.

RPU Seminar 2016

Fri, 24 Jun 2016 00:00:00 +0000

Available Downloads

Get the slides in PDF format.

Download PDF

Unicorn 7 user separation requires manual intervention in a network user environment

Mon, 09 May 2016 00:00:00 +0000

Since we are operating our Äkta Avant in a multiuser environment, we need to separate the methods and results of the different users. By default, every network user is at the moment able to see every method and every result that has been generated on the machine by any other network user. This is a considerable privancy and security issue as a malicious user could delete (or even worse: modify) methods and results.Our Äkta is set up in a way that allows users to log into the Unicorn 7 computer with their university login/password via the regular Windows network authentication mechanism. However, if several people share the responsibility of a run, this setup becomes impossible as they would need to devulge their passwords to each other. Hence we have created a local account which can be used by users who wish to share the operation of the Äkta.After logging into Winodws, users still have to log into the Unicorn 7 software, which users do with their university login and password (“Windows authentication”). Using this setup, every user is able to see all methods and results, which is not acceptable.When setting up a new user with Unicorn version Access>Folders") and exactly which folders were accessible by that user and the user would see only his/her own methods and results. Since the folder structure under Unicorn 5 was a folder structure of the Windows file system, users could always copy methods and results from one folder to another and thereby make them available despite the limitations set by the Unicorn 5 program.When I first read that Unicorn 7 supports Windows network authentication, I had hoped that we would be able to avoid the painful user setup which we had to go thru for each user on the Äkta Explorer. However, the pain continues as setting up the user privileges once for a group doesn’t give us user isolation.Firstly, one cannot restrict access of individual users in Unicorn 7, but only access of groups. We had to create one Access Group for each network user, add the network user to this group, create a separate home folder for the group and then restrict the folder access to this home folder. Hundreds of clicks were required for a handful of users since the default is no access to anything and every single privilege check box needs to be enabled except for the admin privileges.In that respect, Windows (and every other OS) is way smarter than Unicorn. If a university employee logs into a machine that he or she has never been logging into before, it creates all the necessary default local folder structure automatically and mounts that users private home folder without granting access to everything other employees have been doing on that specific machine. I think that should be an option on Unicorn as well. Maybe it is and I just can’t figure it out?Another big drawback of the above described method of separating each user into an own access group is that login into the Unicorn program becomes a major ordeal: In addition to writing user name and password the user has to select the correct access group for the login to be successful. And even worse: In our setup we cannot avoid that every university employee belongs to two access groups: A manually created access group for each user for user separation and the “default” which works via the Windows network authentication - maybe Kerberos?). Hence, if users do choose the default access group (which is called “Users” in our case), they are able to log in, but they don’t see their methods and results.There are two reasons we cannot delete the “Users” access group: One is the mandate of the faculty and secondly (and we have tried), we cannot delete it anymore as many people have already created methods and generated results being in the access group “Users”. Thus UNICORN prevents us from deleting this account. I am working on this problem: I should be able to access directly the underlying MS-SQL database in order to change the ownership of the methods and results. However, GE was not exactly forthcoming when I was asking about access right handling. The answer was:The DB access credentials in a standalone UNICORN solution are encrypted and are not public. If you had an enterprise solution (hosting your own (SQL server) DB) you would have control of the credentials and in theory you could extract the wanted information (the format is something that you have to figure out by yourself and is not supported by us). You can upgrade your solution to an enterprise if you want.This sounds worse than it is, because we have physical access to the MS-SQL server and pulling out the access credentials seems not very difficult. But it takes my time to find the exploit to “break into our own system” and that is what annoys me. However, according to Lisa Bromark from GE, the 7.0.2 update seems to correct this issue:UNICORN can be configured to use a new database password. It is possible to generate an encrypted password or to enter an already encrypted password. This is done by running the UNICORN Service Tool after UNICORN installation.However, it is unbelievably difficult to get the update (at least it seems to take weeks). Distribution is apparently still via optical media and snail mail. I think the last time I got myself software via a CD/DVD was more than 10 years ago. However, GE told me that they are just moving UNICORN software updates to “electronic distribution”. Welcome to the 21 century!

The logic of the Äkta Avant fraction collector

Mon, 02 May 2016 00:00:00 +0000

After we solved all the [teething troubles with our Äkta Avant]((/en/akta_avant), we finally dare to let customers use it. Our customers are very heterogeneous covering complete novices to FPLC and experienced Äkta Explorer users (and everything in between). When we have been giving feedback to GE, our perspective is obviously biased towards a certain type of user. However, taking care of customers is giving us now a new perspective since we get confronted with the usability problems that they cannot solve by themselves. Here I just want to mention one stumbling stone, that has repeatedly brought up to us: the rationale behind the operating mode of the fraction collector. Unlike in the older systems, the fractions collector cannot be manually reset to “First position” or to any arbitrarily defined position as was possible e.g. under Unicorn 5.It took us ourselves quite a while to get used to the internal logic of the fraction collection process and we needed guidance from GE. The fact that the system is not behaving intuitively is underlined by the fact that some of the answers that we received from GE experts were incomplete (leading for us to some unpleasant sample losses). Finally we received from GE support a table that describes the behaviour of the fraction collector (see below). However, even that table is incomplete and we have added a few lines that describe some non-standard situations for which the table does not provide an answer. These changes and additions to GE’s description have been marked in red.

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).

Review of GE Healthcare's Äkta Avant 25

Wed, 02 Mar 2016 00:00:00 +0000

About one year ago, we secured funding in an internal faculty call to replace the old Pharmacia Äkta Explorer 100 FPLC machine of the Protein Production and Purification core facility . The Äkta Explorer had been purchased in 1996(?) when the Swedish brand Pharmacia still existed. In 2014, the worse case scenerio happened and both monochromator and Xenon flash lamp broke almost simultaneously, which left us with a bill of almost 10000€. Hence we wanted to replace the machine with a contemporary model. There are only two companies offering serious devices in this space, which is GE Healthcare ( Äkta product line ) and BioRad ( NGC product line ). GE Healthcare inherited the Äkta brand from Pharmacia via multiple mergers, while BioRad is a relatively new contender with their NGC models, which they released only a few years back.We opted for the Äkta Avant 25 . We definitely wanted to have a cased, refrigerated fraction collector. And we wanted to have a familiar user experience. We often run automated purifications and we do not like our proteins to be for longer times at room temperature in open tubes into which bacteria and dust from the air can enter.By now, we have operated the Äkta Avant for about half a year and we have meanwhile a good idea how it compares to the Äkta Explorer. Even though the Avant is more modern than the Explorer, all users of our core facility still use the Explorer. They are familiar with the user interface of Unicorn 5.11 and seemingly have no interest in learning the - admittedly - more complicated UI of the Avant. But the other reason it was only in internal use so far was the fair amount of problems that we have encountered. With our old Äkta Explorer, we have never seen so many problems in such rapid succession. I don’t know whether our situation is typical (according to GE Healthcare’s representatives it is not). Nevertheless, here are the issues that we encountered:

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…

SnapGene - Simply the best DNA manipulation software

Fri, 01 Jan 2016 00:00:00 +0000

Our lab has been using different software packages to plan, document and visualize DNA constructs. Among those that we liked a lot for a long time were Textco’s GeneConstructionKit (GCK) and Clone Manager (Professional) . The latter runs unfortunately only under Windows. However, since several of our computers run Ubuntu Linux , we did run GCK versions 2.5 and 3 using WINE (a compatibility layer that allows us to run native Windows programs under Linux). However, with the upgrade to version 4, GCK became unusably slow under WINE and we were looking for a replacement. We contacted the developers of GCK, but they apparently were either not willing or able to help us. I suppose that the codebase of GCK is probably more than 20 years old and for that reason nobody dares to touch it. Just around that time, SnapGene was released and it fulfilled almost all of our requirements:

Neon electroporation device chickens out

Tue, 04 Aug 2015 00:00:00 +0000

UPDATE: We are not the only ones that try to economize on our running costs. This lab published its experiments in with the Neon system in Analytic Biochemistry . Thanks to Joachim Goedhart ( @joachimgoedhart ) for bringing this to our attention! The Neon transfection device from Life Technologies (oops, Thermo Fischer nowadays and former Invitrogen ) was introduced about five years ago to the market. It is designed for easy electroporation of mammalian cells. We have had the device available since 2011, but it was not much in use. I don’t know whether the low adoption rate is due to the user-unfriendliness (I still don’t know how to put the electrode tip to the pipettor despite having done this hundreds of times, it’s just really finicky mechanics), expensive running costs (for its desposible gold-plated electrodes and the proprietary transfection buffer) or something else I cannot figure out.However, there are a few things that I wanted to share because real useful information about the Neon device is scarce on the web.The first thing that we had constant problems with were air bubbles in the electrode tip, which result in desastrously low electroporation efficiencies. The only way to really prevent this is to prepare at least 25% more cell suspension than actually needed. When you prepare only 10% more, the last electroporation will certainly arc due to unaviodable air bubbles (the cell suspension additionally sticks easily to the outside of the pipette tip which contributes to the need to prepare more than actually needed). As a consequence of this, we ran out of electroporation buffer R long before we ran out of pipette tips. Additionally one cannot purchase buffer R separately. The Life Technologies representative with whom I corresponded promised to send us a small batch of pipette tips/buffer in good will (that was in January), but we are still waiting for that to arrive…Unforatunately, recently our old Gene Pulser II electroporation device broke. Bio-Rad doesn’t repair it anymore and also doesn’t provide spare parts. It was mostly used for E. coli electroporation. I knew that the Neon device was not designed to electroporate E. coli. Why not and why can’t it be used for that purpose? It turns out that the electric field strength is by far not enough for E.coli (in the BioRad Gene Pulser II, a typical bacterial electroporation achieves an electrical field of 12.5kV/cm, whereas the Neon barely achieves around 850V/cm. However (I thought), the neon can do much longer pulses than the Gene Pulser II (Neon is advertised to be able to give pulses up to 100 ms, whereas the typical pulse length of the Gene Pulser II is about 5 ms). In addition to this, the Neon can deliver automatically multiple pulses. So I wanted to test whether a long and/or repeated pulse with lower field strength can transform E. coli. However, it appeared that when you use water (or 10% glycerol) as the E. coli transfection buffer (which you usually do), the machine complains the tip electrode doesn’t make contact. This is due to the fact that the machine tests whether you have inserted the tip correctly by sending a small current through the system and that current doesn’t flow if you have resuspended your E. coli in water. In order for the machine to “accept” an inserted pipette tip electrode, you need somewhere between 10 and 20 mM NaCl. So I used 15 mM sodium chloride as E. coli electroporation buffer and set the electroporation parameters to 2500V and 100 ms. Surprise: The machine refuses to give such pulse because it is “Over power limit!”. The maximum pulse length it can deliver with 2500V is 19 ms. Unfortunately even that pulse cannot be given automatically multiple times (again: “Over power limit!”). Therefore I manually executed this pulse between 1 and 20 times, but not a single bacterium received any DNA and all bacterial plates remained blank.The manual states:"The Neon TM device is designed to only input certain values and limits for each value are listed below. If your input value exceeds the maximum value, an error is displayed.Input Voltage range: 500–2,500 VInput Pulse Width range: 1–100 msInput Pulse Number range: 1–10Unfortunately, this can be very easily misunderstood. It was not clear to me that one cannot combine the three parameters within these ranges freely. One should think that Life Technology has better technical writers (but maybe they don’t use the device…)Bottom line: The device is utterly useless for anything but mammalian cells. Also some other interesting applications (e.g. electroporation of nematodes or other small critters) are difficult or impossible. While the machine might be a good choice for many mammalian cells, it’s much more limited than the old-fashioned BioRad Gene Pulser II.P.S.: I used the buffer E to fill the pipette station (for use with 10 µl tips). However, I also tested instead of buffer E a mixture of 90% 150 mM sodium chloride and 10% glycerol (which gives me the same conductivity as buffer E has). However, I still really would like to know the composition of buffer R. Why? Because I think that the tip electrodes can be recycled more often than only twice (other manufacturers of pipette tip electrodes advertise that their electrodes can be recycled many more times (e.g. the BactoZapper , although they don’t give precise numbers either). The Neon manual states that “Oxide formation at the piston surface area can be generated if the tips are used more than 2 times, which decreases electrode function of the piston.” The electode is gold plated and gold should be more resistant to oxide formation than the stainless steel electrodes of the BactoZapper…

2-week Lab Course

Tue, 30 Dec 2014 00:00:00 +0000

I had no idea how much work it is to organize a practical lab course. Had I known, Pirjo would have had a much harder time to convince me to give this course for the Doctoral Programme in Biomedicine (DPBM)l . Kari had warned me… The accompanying lecture course had been running from September to November. The practical course had been offered with 16 free slots, but that was totally unrealistic given that we were confined to my 23.7 square meters of lab space. Teaching lab space is available, but without equipment and all the other infrastructure that is needed for such an undertaking. 8 people registered to the practical course and - luckily - half of those pulled out in the last moment with insufficient possibility to commit to the heavy workload that the course required. Thus we ended up with four students and three projects. Under no circumstances would we have managed with more.The idea was to offer each participant the possibility to realize his own DNA cloning and protein expression project. Something that would be relevant for his own PhD studies. For that matter, I had meetings with the three groups one month in advance to plan the cloning and to order the necessary materials. We were working in parallel on the following three projects:

The case against FastDigest®

Wed, 25 Jun 2014 00:00:00 +0000

Restriction enzymes continue to be important

In 2006, the innovative Lithuanian biotech company Fermentas launched a new product line for molecular biology researchers: the FastDigest restriction enzymes. Together with the polymerase chain reaction (PCR), restriction enzymes (REs) are arguably the most important tools in molecular biology. They made recombinant DNA technology possible in the early 1970s. Until every lab can afford a reliable DNA laser printer (which is probably still a decade away), restriction enzymes are the tools of the trade. Novel cloning techniques (like In-Fusion , Gibson Assembly or SLICE ) have their place, but still lack the robustness of traditional restriction enzyme cloning.

Stripping

Fri, 01 Nov 2013 00:00:00 +0000

Reprobing membranes with with a different antibody is a very common task in the lab. Various protocols exist to strip membranes and the classic method is the one that uses SDS, β-mercaptoethanol and heating. I used to do it that way, but it’s a smelly business, because β-mercaptoethanol smells like rotten eggs. Then suddenly everybody in the lab started to use the Re-Blot Plus Solution from Millipore and so did I. Until I realized by chicking the Material Safety Data Sheet that they sell cheap chemicals for a premium price. Now I make the stripping buffer myself. My 10x solution has the following composition:

Recombinant proteins

Fri, 25 Sep 2009 00:00:00 +0000

A dynamically updated list of proteins used to be here, but I shut down the communication to our lab’s database server due to security concerns.