Purify by homogenizing in 0.25M Sucrose, 50mM HEPES, 1mM MgCl2 with protease inhibitors. Then do a sucrose gradient using an ultracentrifuge. You can look up how to purify microsomal cytochrome P450 to determine how to make the sucrose gradient. Take the layer that has your activity or proteins and then depending on what you want to do, you can solubilize with detergent, and then do standard chromatography such as ion exchange and affinity chromatography.
What you do, depends on what proteins you are trying to obtain, and if they can survive in detergent. The detergent is usually non ionic like NP40 or DDM. Sometimes Triton X 100 is used. The detergents need to be tested empirically to see which one is best suited for your needs. And the best method depends on your protein as well.
thank you very much Marcia, appreciated, can you please send me the affinity or ion exchange chromatography protocol, thank in advance
Ion exchange
If you know the PI, or isoelectric point, the use a pH where the protein will have a net positive or negative charge. You will know this because histidines have pKa around 6.5, carboxylic groups, pka around 3-4 and amine groups around pH 9-10.
So if your PI is near neutral, then dropping it to around 6 to create positive charges because of histidines would make it stick to the SP resin, for example. SP resin is negatively charged.
If you don't know the amino acid composition of your protein, then I empirically try different procedures. If you don't have fancy equipment, then start out at neutral pH and see if your protein sticks to cation or anion exchange resin. I use Q sepharose ( the Q stands for quaternary amine so it binds things that have a net negative charge) or SP sepharose ( the residues are negatively charged). You can use any resin for ion exchange that has positive or negatively charge residues.
If it does not stick, then change the pH up or down and repeat.
Once you know it will stick, then prepare a column with the resin of choice. Equibrate in your buffer system, ( it should be low salt so that it will bind the resin) and then load it, wash the column, with low salt, and then run a gradient from 0 to 1M NaCl using 10 column volumes. Collect fractions and find your protein.
If it does not like to stick to anything, you can still pass it through a column at low salt, because other proteins will stick to the column.
Affinity chromatography
It depends on your protein.
1. If it has glycosylation, then use conocandin, A, wheat germ agglutinin resins, etc. elute with sugars like D mannose.
2. If it is hydrophobic, use phenyl, hexyl, or octyl sepharose chromatography. Load in high salt, and go to low salt. You may even have to go no salt with glycerol.
3. If it binds certain cofactors, then find resins that have the cofactor bound to it, or use pre activated resins to attach what you want to the resin. For example there are ATP columns, and co acorn blue binds proteins with NADPH, etc cofactors.
4. If it binds an inhibitor, agonist or antagonist or other protein, you can biotinylated these and purify with streptavidin resin. ( as an example) or you can attach directly to the resin as in (3). See Pierce catalogue for some options.
hi,
This the best to guide for me.
it`s sincerely appreciated.
Thank you very much Marcia
Have a great day
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