why do we do each step?
1-Let the eluate flow from the column until the level of buffer almost reaches the column
carrier material. The column carrier material should not become dry, so close the column
with a clamp
2- Before you start purification of the GFP sample measure the protein concentration of the
supernatant using the Nanodrop,
3- Apply all the supernatant of your GFP protein isolation (with 1.5 M (NH4)2SO4) to the
column, open the clamp, wait until the fluid level of the extract and column carrier
material is equal and closes the column with the clamp. Still collect the eluate in a 100
ml beaker(= waste).
4-Apply buffer 1 to the column in quantities of 1 ml and let it be adsorbed (without
entrapping air bubbles); repeat this five times and check whether GFP is on the column
(with UV-light). From this moment on (start of applying buffer 1), the eluate is collected
in micro-centrifuge vials (+/- 1 ml per vial).
5-Now replace buffer 1 by buffer 2 (= 10 mM Tris-HCl pH 8.0 )
step 1: after equilibrating the column, you should leave a small amount of liquid at the top so the column does not dry because if it does, the beads may break and the column resin will crack apart giving you a discontinuous resin bed.
step 2: one should always know how much protein is being loaded onto a column. Each chromatography resin has a maximum amount of protein that it can bind (i.e. total protein per mg resin) so you need to make sure you have enough resin to handle the protein loaded. you can use nanodrop or other methods to measure protein concentration and total protein to accomplish this. This is helpful because you will know your recovery rate (yield) and hence degree of purification of target biochemical- and perhaps know that some protein is still attached to column after last step.
step 3: after loading the sample, stopping (clamping) will assure that all loaded material has a chance to bind with the hydrophobic resin. It is always good practice to save everything that is eluted from the column in case there is some error, no sample is lost.
step 4: adding 1 ml amounts of buffer in several successive steps and allowing to enter the resin helps push the loaded chemical down the resin slowly and most importantly prevents the biochemicals from ending up on the buffer on top of the column. this repeated additions of buffer assure that the loaded material moves down into the resin. For instance, it is possible that after the first or second 1 ml addition of buffer, some of the chemicals at the top of the resin may mix with the buffer (since the top of the resin is disrupted as you add liquid) and are then in the liquid part and no longer in the resin. That is why it is important to let it all go into the resin before adding the next 1 ml, and so on. Of course, one should be collecting the eluate, as always, to make sure the target biochemical is not lost.
step 5: at this point the proteins that bind the resin need to be removed, and hopefully those that did not bind (and are the ones that you do not want) will have eluted. By changing the buffer, you disturb the hydrophobic interactions and your protein will elute. If it is not the last step of purification, other proteins that bound to the column resin will elute as well.
good luck!
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177899/
https://www.ncbi.nlm.nih.gov/pubmed/21968976
https://www.gelifesciences.com/en/us/solutions/protein-research/knowledge-center/protein-purification-methods/hydrophobic-interaction-chromatography
Thanks
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