Hello, i purify a different proteins from bacterial culture using gel filtration technique, i have 2 types of gel sephadex G20 and G100, i want to know:
1- the dimensions of the column i should use.
2- the concentration of the gel i should prepare.
3- the amount of gel that should be added into the column.
and finally 4- the flow rate.
thanks.
This manual is very instructive.
http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc18102218_20141208003707.pdf
It depends a lot on your sample size. For sizing columns, one uses about 1ml per every 100 ml of resin, and you want to use as long a column as possible. As an example, I used about a 2 inch diameter, 30inch column, with about 300ml of resin, and this gave good separation between 20kDa and 40Kda proteins using G75.
Length of the column is directly proportional to the separation of protein.
If you use G-20, it will give better separation in low molecular protein. In the case of G-100 will give better separation in mid.
Conditions:
G100 Sephadex
1 m height, if possible (longer, better) (Diameter 1-2.5 cm)
Low flow (5-10 mL/h)
1 ml fractions
UV (280 nm) protein detection in fractions.
G20, is too risky because exclusion (no separation) of medium and high MW molecules.
Yours,
Hector
Besides planning for the sample volume to be 0.5 to 1% of the total volume of packed resin in the column, and favoring height over diameter in achieving that column volume as recommended by others, I also agree with not going smaller than 1 cm in diameter while still experimenting with conditions. I would also add that you might want to try to concentrate your sample to a small volume. You will be better able to detect the minor peaks and also reduce the size of the column that you will require. But do make sure that the process of concentrating the sample did not cause some protein to precipitate out.
for gel filtration, what you need is a long column sufficiently enough for separation, sufficient flow rate to avoid diffusion (longitudinal or eddy). I had purify proteins in analytical column (size of an HPLC column), normal column (GE or Biorad), and large one that its length is about 1.5 meter (self pack). All worked well. From my experience, the width to length should be about 1:20 ratio (1 cm diameter to 20 cm length). For such dimension you need 3.14 x (1/2)^2 x 20 ~ 15.5 cm^3, thus about 15-16 ml. you can increase the ratio up to 1:30 but I don't recommend you to have the ratio too large, because you will start to experience diffusion at whatever flow rate (especially if your run is based on gravity).
From your question I assume you have resins, not column? The most important thing is to have a good homogenous distribution of resin in the column. Otherwise, no matter how ideal your column dimension is, you will never get good separation. so, good packing is crucial. Good luck.
- Badges
- Science method