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Fusion Protein purification using G-column, but really poor results

antibodies Affinity Chromatogr fusion protein

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#1 azt129



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Posted 16 May 2012 - 02:12 AM

Hi All

Its my first post, but here goes

We are currently using 293k cell lines transfected to express a fusion protein we plan to use as an antibody (or marker)

We received a protocol from another group that had been successful in doing so:

1. Plate cells in 4 500ml flasks with 50ml medium

2. Grow the cells in the flasks until they are adherent, but not confluent. (when the cells begin to cluster, contact inhibition impedes them from secreting the proteins.)
3. Remove the medium and replace it with 200~250 ML Low Protein Medium without FCS.
4. When the medium turns orange~yellow, but before the cells start to die, collect the sup in 50 ml falcons. Centrifuge, pool and filter. Add 0.05% Soduim Azid.
5. Standard G-coulmn protocol to elute protein
6. The proteins should be in a 7~8 pH environment so that they won’t degrade.
7. Transfer each fraction to a dialysis membrane and stir them in a container of 2 Liter PBS over night. Repeat this with new 2 L PBS.

Measure the protein concentration (should be anything between 01 – 4 µg/µl)"

We followed the protocol pretty closely but used 15cm dishes instead and had about 11 dishes. We found we only got around 0.08 µg/µl at the end, which was far too low. Even before the dialysis, we only had about 0.13 µg/µl

After a bit of brainstorming we decided ways to improve the concentration to this protocol may be:
a) use less medium per plate
B) perhaps plate more dense (they were at 60%)
c) elute to smaller fractions
d) grow cells for longer period in Low protein medium w/o FCS?

The problem was we didn't know what density we should be plating at, so we will be doing an experiment to try and find to optimal density.

I had also wondered about the cell culturing part, perhaps culturing in lower levels of essential amino acids beforehand, and just prior to switching to Low protein medium w/o FCS, add more essential amino acids, hopefully boosting protein production.

Does anyone know of any other ways to promote protein production/secretion from cell lines that may be appropriate here?

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