SEC5K – Purification Column
The Nanovex-SEC5K allows quick and economical purifications (protein, nanovesicle or nucleic acid) with exclusion limits of 5,000 Daltons (components larger than 5,000 Daltons eluted with void volume). The Nanovex-SEC5K columns are shipped containing ultrapure water/ethanol (20% v/v ethanol) as a preservative.
- Shake the column and its contents gently several times, in order to resuspend the resin. Then, place the column upright and allow settling the resin bed.
- Carefully remove the storage solution from the top of gel bed.
- Equilibrate the column by adding five gel bed volumes (50 ml) of the desired buffer.
Sample purification or desalting
- Carefully remove the buffer solution from the top of gel bed.
- Add the desired sample volume to the top of the column and collect the same volume of eluent (fraction 1). After the sample has entered into the column and the first fraction of eluate has been collected, add a volume of 1 ml of the buffer solution, and simultaneously collect the same volume of eluent from the bottom of the column (0.5 ml fractions) as they emerge from the column.
- Repeat this step until the desired number of fractions has been collected.
- Then analyze the collected fractions with the appropriate detector and plot the recorded signal against fraction number.
- Removal of free low molecular weight molecules from protein
- Removal of free low molecular weight molecules from nanovesicles.
Example 1: Separation of nanovesicles containing fluorescein from unencapsulated fluorescein.
- Equilibrate the SEC column with five column volumes (50 ml) of PBS.
- Apply the reaction mixture (200 µl) to column. When the reaction mixture has entered into the column and the first fraction was collected, add PBS and continue collecting separate 1 ml fractions as they emerged from the column (Typically 30 fractions are collected).
- For detection of nanovesicles and unencapsulated fluoresceín, absorbance measurements at 500 nm wavelength of all fractions were carried out (Typically fractions 4 to 6 will contain the nanovesicles, see the figure below).
Example 2: Separation of protein modified with SATA from unincorporated SATA.
- Equilibrate the SEC column with five column volumes (50 ml) of PBS containing 10 mM EDTA.
- Apply the reaction mixture (1 ml) to column and collect 1 ml fraction. When the reaction mixture has entered into the column and the first fraction was collected, add PBS solution containing 10 mM EDTA and continue collecting separate 1 ml fractions as they emerged from the column (Typically 10 fractions are collected).
- Protein detection is performed by absorbance measurements at 280 nm of all fractions. (Typically fractions 4 to 6 will contain the protein, see the figure below).
|Column Volume||12 ml|
|Sample size||Up to 1 ml|
|Volume gel bed||10 ml|
|Void volume||˜3.3 ml|
|Bead diameter (wet)||85-260 µm|
|pH stability||2 to 13|
|Autoclavable||121ºC, pH 7.0, 30 min|
|Chemical stability||All commonly used buffers, included:|
1M Acetic acid
Store in a cool and dry place and protected from the light.