Dear colleagues,
I am trying to purify a app.11 KDa protein (Enzyme) with chromatography method. I am going to use superdex G75 resin with a column length of 60 cm and 120ml of resin.
but i do not know how much protein do i need to load in the column. can anyone help me with that. ? ?
i have already purified my protein with Ni-NTA method and have an almost pure protein of interest. i can say 90% purity.
thank you all in advance.
Keep the volume of protein as low as up to 0.5 mL to 2 mL and up to 10 mg/mL protein.
best wishes
Dear Ehsan:
Most of the companies providing Superdex G75 resin suggest to use samples containing less than 10 mg of protein in the sample. Here you have some links with more details:
http://research.med.helsinki.fi/corefacilities/b3p/manuals/columns/Superdex75and200_10-300GL.pdf
https://www.gelifesciences.com/en/bw/shop/chromatography/prepacked-columns/size-exclusion/superdex-75-10-300-gl-and-5-150-gl-p-05899
Your column seems to me to big if it is not the last step in your purification process.
Good luck!
Keep the volume of protein as low as up to 0.5 mL to 2 mL and up to 10 mg/mL protein.
best wishes
Dear Ehsan,
usually the supplier provides the information for the maximum capacity of total protein load (depending on the volume of the column). However, a general rule is to not exceed 2% of the column volume for size exclusion, i.e. in your case 120ml --> 2.4ml.
Hope this helps
Use s concentrated protein sample in small volume (say 0.5 to 1 ml) to load on the bed surface for best chromatogram profile. Narrow sharp peaks will be obtained in this case.
have a look at scopes r.k book on protein purification,principle &practical
Diameter of the column (cm) = (m/10)^(1/3)
where:
m = mg of protein.
Length of the column = 30 * Diameter
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