Bio-Rad on Michael’s Domain

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

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:

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…