I was looking for any recommendation for protein purification system for my research. We dont have much budget for akta and are looking for affordable alternative. We do have HPLC in the lab, can we attach column to the machine and purify our protein successfully?
Hi there :)
There are way cheaper alternatives to expensive hardware like Äktas. Often small plastics or a small magnet are enough.
Column Chromatography, Batch Spin, Drip Column or Magnetic beads.
The first 3 use the same purification beads (agarose) as an Äkta would used, while Magbeads serve the same purpose, but you need a small magnet for them:
THis Youtube Playlist shows the methods I mentioned:
https://www.youtube.com/playlist?list=PLgKx7UXRO02v5sXoqfl0_czldMa03Pw0g
It also includes FPLC for Comparison.
Depending on the type of chromatography you want to do, there may be simple, inexpensive methods that do not require sophisticated equipment. The results will not be as well-documented as they are with an FPLC.
For atmospheric pressure chromatography, you can pack suitable resins into glass columns (e.g. Bio-Rad Econo columns) and use a bench-top peristaltic pump to control the flow rate. Fractions can be collected by a stand-alone fraction collector, or by hand if you have the patience. To make a gradient of eluent, you can use a gradient former, or even just two beakers with a tubing siphon between them, and a magnetic stirrer. A stand-alone UV detector can be connected to the outlet, or you can measure the absorbance of each fraction with a spectrophotometer, which is available in most labs, or you can do a quick Bradford assay spot test on each fraction to find the peaks.
Protein purification can be done using certain types of HPLC columns with large pore sizes, but the columns that are large enough for preparative scale HPLC are very expensive. You will have to be careful about making sure the buffers you use are compatible with the system's materials.
Dear Fatin Safiudin Fatin
as Adam B Shapiro told, it depends from the kind of purification that you woud like to do.
If you would like to use affinity purifications as IMAC for Histagged proteins or protA/protG for an antibody you can also do it manally avoiding the use of an liquid chromatography since you can perform gravity flow putifications up to 1ml od resin or use a perislatic pump with 5ml FF coloumns for an higher scale, and use your HPLC just for the final sample quality check,
you can have more details about the matherial suitable for this kind of purification at the following link
https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html
regarding the possibility to use your HPLC sistem instead the AKTA, it depends if the system configurations. We are using for example a superdex200 coloumn in an OMNISEC LALS/RALS HPLC. It work well for analitics but not for purification since we do not have any sample fraction collector. If you are able to inject the required volumes, guarantee a proper flow rate and collect the fraction from the colomn, theoretically any instrumenti compatible with standard acqueous buffers are ok.
good luck
Manuele
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