Hi,
There is a way to make the actual enzyme but the post purification step may involve getting an enzyme that is not active as the fplc in the department is not the best.
Does someone have more information regarding this?
Thanks, I will try. If they cost like normal enzymes its ok. But this is fab news:)
What is the purification step you expect to have trouble with? Maybe there is another way to do it. FPLC is not required to do column chromatography; you can also use gravity columns.
I am a little skeptical about FPLC as I have not done it before and there is no great facility available for it in my department. The protocol involves the use of Sepharose SP columns, is there an alternative using a gravity column for this?
Ion-exchange chromatography with no equipment other than a column and a ring stand:
The procedure should be carried out at 4oC if possible.
Suspend a bit more than the required amount of SP-Sepharose in the starting buffer in a beaker or tube, let it settle, pour off the supernatant, and repeat a few times to equilibrate the resin with the buffer.
Pour the resin into an open-top glass chromatography column (e.g. Bio-Rad Econo column) attached vertically to a ring stand and with a stop-cock at the bottom (Econo-columns come with a stop-cock) and let the resin settle. Drain the buffer until the top of the column is just exposed. Close the stop-cock. Don't let the column run dry.
Gently apply your sample onto the column with a Pasteur pipette or plastic transfer pipette. Avoid disturbing the resin. Let the sample drain into the column. Wash the column with a few column volumes of the starting buffer. Collect the eluent from the bottom of the column in tubes.
Eluting the column with a salt gradient is possible, but a bit tricky, so you might want to use salt steps instead, i.e. eluting the column with equal volumes of buffer containing incrementing salt concentrations. The jumps in concentration should be small, such as 10 or 20 mM at a time. Let each salt solution drain to the top of the resin before applying the next one, to avoid diluting the salt. Collect each salt step as a separate fraction.
When the chromatography is done, mix each fraction so the protein is evenly distributed. Take a sample of each fraction for SDS-PAGE, UV absorbance measurement, or protein assay to measure the protein concentration and find the fractions with your protein in them.
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