I have extracted total protein from a growth medium and I need to split the sample into two fractions - one with all the large proteins and another with all the small proteins (the large proteins are too immunogenic and drown out the small ones so the mice only generate antibodies against the large). The protocol I've been given is a 10% - 40% sucrose step gradient, but this protocol is a real pain. Is there a better way to do this? Any suggestions would be much appreciated, thanks.
hi
you can test " Amicon® Ultra 15 mL Centrifugal Filters " to separate and concentrate your proteins with 3 , 10 , 30 , 50 or100 kD filter
https://www.merckmillipore.com/INTL/en/life-science-research/protein-sample-preparation/protein-concentration/amicon-ultra-centrifugal-filters/15-ml/Q5ab.qB.pKIAAAFBuLplvyxp,nav
good luck
Try to perform gel filtration fractionation using size-exclusion resins with high resolution. Ultrafiltration is also a choice depending on the MW ranges of your proteins.
Thank you for your answers :) I haven't done gel filtration fractionation before. Would it involve FPLC or HPLC?
You don't have to hold an LC system. It is certain that they make it very practical and they pose ease of method development. Alternatively, you may pack a column with SEC moiety (e.g Sephadex, sepharose beads) and perform gel filtration column chromatography in your lab. Load your sample after conditioning and elute your proteins respectively. Check the MWs of your fractions using SDS-PAGE and identify the correct fractions for your purpose.
Proteins have all about the same density, so if you want to separate them by ultracentrifugation, you can't do that isopycnically, but only by rate zonal centrifugation. This is done not on a step gradient, but on a shallow (say, 0-10%) linear sucrose or glycerol gradient. You can read about ultracentrifugation in books on physical biochemistry like my own.
As others have pointed out, SEC can separate proteins by size. You need a column about 60-100 cm long and wide enough that sample height is at most 1% of the column length. Since SEC is done isocratically (no gradient), you do not need much equipment, a peristaltic pump is nice, but gravity feed suffices. If you don't have a photometer, add dextran blue to the sample to mark the front (high mass end), and a dye like phenol red to mark the tail. Then collect everything between the blue and red band, in, say, 10 fractions. Check the separation by electrophoresis. Again, textbooks like my own will cover the theory.
Hi Catherine,
I am not really sure about what you mean with large resp. small proteins, but as suggested earlier you can use size exclusion chromatography (SEC). Have in mind that there are several different kinds of SEC resins on the market with both different pore sizes and bead sizes and it can be a challenge to find the right one to the best price. If you contact me, perhaps I can help you if I get more details.
Best regards, Anna
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