Fast Protein Liquid Chromatography (FPLC)/ HPLC - (Oct/21/2008 )
Hi,
Is there anyone using Fast Protein Liquid Chromatography (FPLC) here? Is it the best method to separate enzyme? How about HPLC? I am starting my experiment soon.. but I m still blur about the method.. HPLC or FPLC? Please leave some suggestions.
Thanks buddies..
just saying fplc or hplc doesn't mean much. what separation type are you looking at?
fplc (sometimes called mplc) is just hplc with a lower pressure so that the stationary phases aren't subjected to pressures that they can't withstand.
do you want to separate by size (mw), charge, hydrophobicity, hydrophilicity, affinity,...?
fplc columns (and phases) are designed for protein/peptide work but that doesn't mean that you can't use hplc phases for them (and sometimes more effectively).
be more specific about what it is that you wish to do and we may be able to help you decide which may be better for your purpose.
Is there anyone using Fast Protein Liquid Chromatography (FPLC) here? Is it the best method to separate enzyme? How about HPLC? I am starting my experiment soon.. but I m still blur about the method.. HPLC or FPLC? Please leave some suggestions.
Thanks buddies..
do you have the choice? important are the columns/matrices you have; dependening on the protein you need at least 3 different columns for protein purification
fplc (sometimes called mplc) is just hplc with a lower pressure so that the stationary phases aren't subjected to pressures that they can't withstand.
do you want to separate by size (mw), charge, hydrophobicity, hydrophilicity, affinity,...?
fplc columns (and phases) are designed for protein/peptide work but that doesn't mean that you can't use hplc phases for them (and sometimes more effectively).
be more specific about what it is that you wish to do and we may be able to help you decide which may be better for your purpose.
Hi,
Thanks for the reply.. I need to separate the enzymes by size. I m looking for chitinase, beta galactosidase and xylanase. My colleague said its better to use Fast Protein Liquid Chromatography compare to HPLC. This is because, it seems HPLC is suitable for purification. The quantity of the enzyme is very little in HPLC. So i just need to know using HPLC or FPLC is best to separate enzymes. So I m in confusion right now.. I am not sure which one is the best to separate my target enzymes. As a beginner i really need someones suggestion. Hope to get a reply soon.. Thanks in advance.
Abby05
in general, the hplc columns will be useful for lower amounts of material than fplc columns (unless the hplc columns are preparative) and will give better (finer) resolution than fplc.
fplc columns are semi-preparative. you can use them to purify greater amounts of material but you will suffer a loss in resolution. if your proteins are far enough apart in size and/or charge then that won't make a significant difference.
you can also adjust running conditions to maximize the resolution.