I want to separate my plant protein sample via gel filtration prior to affinity chromatography; please guide
The manual is all over the internet, look for it. It will depend on what column do you have.
First: are you sure you want to start a protein separation by G-75? If your sample has too many proteins, you may want to begin with a fractionation with ammonium sulfate or an ion exchange column since in most purification methods, it is not recommended to begin a purification with size exclusion chromatography.
Secondly, is this the right size exclusion column? this one separates proteins between 3K- 80K well but not larger ones.
The first step in preparing a gel filtration resin is to calculate the amount you will need based on the amount of protein that will be loaded. Then, swell the dry resin in an excess of your buffer and allow enough time for swelling of the beads, equilibration of resin at your buffer's pH and remove fines after each swelling step. When packing the resin into the column, close the outlet and let it settle naturally to avoid convection. All other information is in the manual- I just discussed the important points and tips.
Good luck.
Dear Gustavo,
My task is to purify an untagged protein from transformed plants. It's a hexameric globular proteins and it's monomeric unit is of 24 kDa, the pI of protein is 6.4
I have started the fractionation via ammonium sulphate, afterwards I will do a gelfiltration via G 75 column after purification of dialysed protein fractions. I have extracted my proteins in 20 mM Tris HCl buffer pH 7.4 and 50 mM NaCl.
After gel filtration I will be doing affinity chromatography for purifying this protein,
Is it ok to swell the G75 column in the extraction buffer or shall I use 20 mM NaCl, or their is any other specific buffer system
Sorry can I use 20 mM Tris HCl only in preparing G 75 column or shall I use the same buffer as in extraction of proteins 20 mM Tris HCl and 50 mM Nacl
Swell the G75 resin in the 20 mM NaCl with 20 mM Tris-HCl, pH 7.4 to keep the protein 'happy' (i.e., folded and active). The protein should be dialyzed in the same buffer as the equilibration buffer (which is also the swelling buffer). Check the pH to make sure the column resin is equilibrated. Some people also check the conductivity to ensure correct NaCl concentration or to make sure all Amm SO4 has been removed.
Run the column at a slow rate (depending on column size). Ideally in a cold room (4 degrees C) and collect fractions with a fraction collector. Again the volume of the fractions and rate depend on the size of the column.
When allowing beads to swell, decant the equilibration buffer several times to remove fines (broken pieces of resin) which clog the column and interfere with flow rate.
One more thing: if the protein remains as a hexamer, then 6x24K =144,000 and G75 will not be the best column for size exlusion chromatography since it only goes up to 80K. it would be okay if the protein ran as a monomer, but I do not think it will, unless you have done something that would have broken down the hexamer to monomers.
Good luck.
Ok I will give it a try, further I will also do cellulose phosphate cation exchange column for initial separation, I have this in exchanger only.
I will operate the columns manually as I don't have fraction collector
Dear Gustavo,
Will sephadex G 100 will be fine with a fractionation range of 4000-150000
Yes, S G-100 should work fine. Remember that the ratio of loading volume to resin volume is critical. loaded sample volume should be about 5% of the bed (resin) volume to give you good separation. Make sure entire sample is in column before beginning the buffer addition.
Here is a link, in case you are still having trouble with the fine points of equilibration/ column preparation:
http://legacy.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11520&catalogId=10101&productId=21028&storeId=11787&langId=-1
Best,
G
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