ProteoCool Pills#16: Loading small sample volumes on AKTA instruments

AKTA instruments (as AKTA-FPLC, AKTA-PRIME, AKTA-Purifiers, AKTA-Go, AKTA-pure) represent in the last 30 years the point of reference for protein/antibody purification in both research, development and pharmaceutical production departments since they are robust, easy to use and provided with a software (the Unicorn)  which is a reall uses friendly software.

On the contrary, their application for protein/antibody analytics characterization is mainly limited to some specific columns and approaches (eg SEC chromatography with the Superdex 10/30 or 5/15 coloumns) in those laboratories that do not have budget dedicate to acquire an analitical HPLC. 

 In those case the loading and injection of a small sample volume in reproducible way it a prerequisite.

How we can inject small volumes in an FPLC or other AKTA instruments? 

For volumes between 200-300µl to 1ml,  the standard injection port could be used with an 1ml insulin plastic syringe (with out needle)

For volumes <250µl ,  the standard injection port need to be replaced with a injection filling port designed for small volumes and use an more precise Hamilton syringe (250µl or 100µl volume) for sample injection.  

Until some years ago (as I already wrote to answer to a question in Research gate in 2019) https://www.researchgate.net/post/Loading-small-sample-volumes-on-FPLC), Citiva bioscience (previously GE bioscience) supplied for small sample injection a metal injection filling port (CITIVA cod. 19768701) supplied with a plastic syringe stand suitable for injection of small volumes using Hamilton syringes

Recently those Injection fill port was discontinued and replaced by a plastic filling post (Fill Port, INV-907 cod.18112766)

Also if this filling port, is not supplied with any syringe stand, it could be used directly with an hamilton syringe, as you can see from the following imagine:

Of course to inject so small volumes you need to select also the appropriate:

•          Injection loop (with a small volume)
•         Syringes (with BLUNT tips:

Suggested:

50-100ul sample:  Hamilton 1710N volume 100 μL, needle size 22s ga, needle L 51 mm; Sigma-Aldrich cod. 28634-U cost ~100€;

250-100ul samples: Hamilton® 1725N, volume 250 μL, needle size 22 ga (blunt tip), needle L 51 mm; Sigma-Aldrich cod. 28636-U cost ~80€;

 

 

ProteoCool Pills#15: An alternative way to fix the sticky pad to the Eppendorf S41i CO2 shaker

In the last 20 years, thanks to the adaptation in suspension of several cell lines and to the development of more efficient transfection agents (eg lipofectamines) and culture media, the scientist were able to obtain amazing progresses in the performance of mammalian expression systems, 

As I already reported in the ProteoCool n° 29 and in the tips of the week#8 , modern cell lines, as  ExpiCHO and Expi293, are able to produce high levels (up to 300-500mg/l) of recombinant antigens and/or antibodies in the culture surnatants also in shaking flask culture. 

The availability of an orbital shaker able to maintain 5-8% of CO₂ atmosphere is essential during cell propagation and maintenance.

Differently to bacteria, the cell lines do not require high shaking rates (generally 120-125rpm) but are more prone to contamination, therefore since the cleanliness is essential, the use of sticky pads, those can be easily removed and sainted with ethanol or isopropanol solutions is preferable respect than the conventional flask clamps.

To my knowledge Eppendorf S41i (previously New Brunswick) and  the Infors multitron  are the mostly used for mammalian cell cultures.

In this presentation I would like to share with you,  my experience with the Eppendorf S41i

In our lab we have 2 Eppendorf S41i shakers:

- 1st setted at 37°C- 8% CO₂ and 120rpm for propagation of both Expi293 and ExpiCHO and for transfection of Expi293 cells;

-  2nd setted at 32°C- 5%CO₂ and 120rpm for the transfection of the ExpiCHO cells; 

The first shaker was acquired in 2016-2017 while the second in the 2020. 

Our older S41i shaker carry a platforms characterized by a smooth surface and the sticky pads attach well  directly to its surface

                   while our newest  S41i shaker carry a platforms characterized by a rough surface and the                         sticky pads don’t stick well to its surface.

                   Eppendorf sell a Stickpad ADAPTOR KIT which contains 2 metal smooth plates

                                but this kit seems to be designed for a different shaker format since

               the sizes of those plates are not appropriate to cover the entire area of the shaking platform.

Since the Eppendorf S41i is smallest than other shaking incubator, e.g. the multitron, 

 giving up such a large part of the plate surface represent an important limitation that may 

induce the scientist to choose a different shaker for their laboratories. 

In this post, I would like to show how we were able to cover with the sticky pad the entire shaking platform of both our shakers

           You can use the fixing screws supplied with the conventional flask clamps or wtih the Stickpad ADAPTOR KIT  to drilling and fixing the sticky pads directly on the black  shaker platform.

Standard Washers with 6mm in diamteter

 those can acquired in the most hardware stores /e,g  Leroy Merlin, Obi, Bricoman, Brico ) to ensure the sticky pad integrity and extend the life time of this solution.

N.B I'm happy to share with you that, as responce to this post, Eppendorf specify  that all the platforms actually produced are chatacterized by a smooth surface and the sticky pads are able to attach directly to it.