I am trying to purify extracellular enzyme from culture supernatant, for that reason I concentrated SN and performed ion exchange chromatography using manual method (NaCl gradient in buffer, pH-8). Fractions I collected, measured absorbance at 280nm to detect protein content (which didn't show 2 different peaks), checked enzyme activity and later concentrated enzyme containing fractions. This fraction when subjected to zymogram analysis I found 2 active bands which have close molecular weights (close pI values also, since both eluted in same fractions).
Hi, Pankaj,
When you say 'close molecular weights', how close is this? Maybe there is a chance to separate them by size-exclusion chromatography on the right support and under some optimized elution conditions (low flow rate at least). Of course it could be that the two active bands you see are two isoforms that are actually one protein with different posttranslational modifications, which could be tricky to separate on preparative scale. You might consider hydrophobic chromatography, but eventually a well-selected IEC support, pH and gradient design could do the job as well. It might help if you provide your exact chromatography protocol and any photographs of your current results.
Thank you very much sir, I'll try to work on size exclusion.
My both protein bands on SDS-PAGE appear in between 31kDa-45kDa, which is quite narrow range but as per your suggestion I'll try to separate them by controlling flow rate.
The size difference you mentioned (~10 kDa) is sufficient to separate the two proteins by SEC unless they stick to each other or some other proteins in your samples. Sometimes PAGE gives us a wrong idea about the actual state of the protein of interest. Keep us informed about the progress.
I think Marko Dolinar has the right idea. Simplest thing to do if your proteins are very similar in structure/properties is to use size exclusion chromatography. I just wanted to add though that you will likely need quite a long column to effectively separate these proteins in any significant quantity. Just some suggestions in this regard to get you started:
1. Use a column that is quite wide. Normally a much smaller column could accommodate the protein loading but I have had good luck with using a much wider column than "recommended" as this means the proteins will migrate as a thinner band/peak since they can spread out laterally more.
2. Use a long column. You may even wish to connect two columns together if you are able in order to increase the resolution but be sure to use a very low flow rate to ensure you don't overpressure the first column.
3. Make sure you use a column that has the right separation range.
4. As Marko Dolinar mentioned run the column slowly, its ok to run it at a very gradual flow rate overnight provided you have a fraction collector to collect the eluent
Overall I would recommend something like the GE HiPrep 26/60 or 16/60 S-100 HR. Another configuration which I have used in the past would be to run them through a GE Superdex 75 10/300 GL and connect the output of that column directly to the HiPrep 26/60 or 16/60 S-100. The 75 10/300 is a narrower column to the peaks coming off are a bit broad but it will begin the separation, once the peaks hit the S-100 they will start migrating faster so that will boost the separation. It's important that the 75 10/300 go first though because it has a higher pressure rating and lower exclusion limit. The pressure rating is important because if you put the column with the higher pressure limit/resistance after the column with lower resistance you can overpressure the first column. Putting the column with the higher exclusion limit second is also important because then proteins will run faster once entering the second column. Putting the column with the higher exclusion limit first would cause proteins to "pile up" when they reach the second column and start running slower.
As Marko Dolinar mentioned, any pictures/data/chromatograms would be helpful in assisting you through the process.
Size exclusion chromatography (Sephacryl or Sephadex matrix for instance) or SDS-PAGE using acrylamide gradients would help you in this task.
Good luck!
SEC would be the preferred method, however constraints of concentration and height of column is an issue. IEX is a last option. Equilibrate IEX in buffer used for binding, then charge sample at 1/2 the binding buffer at same pH, then follow with a 5 x gradient that will pass through the ionic strength that both eluted previously.
Single peak in Ion Exchange chromatography elution does not mean they are of same molecular weight, it is based on Pi. Use a shallow gradient and check still you get a single peak and also you could do a gel filtration column which is based on molecular sizes of the proteins.
Thank you all for your precious guidance and suggestions, I'll update my results following suggestions given by you all.
Marko Dolinar, Rosa María Martínez-Espinosa, Vernon L Reisenbichler, Peramachi Palanivelu, Kou Hayakawa
, Zachery R Belak, Jai Ghosh
Try ammonium sulfate precipitation. Gradually increase the ammonium sulfate in your protein solution until one of your proteins start to precipitate. Centrifuge and separate the pellet and supernatant. One of the protein will be in the pellet and the other protein will stay in the solution. Resolubilize the pellet in your buffer.
If you go on adding ammonium sulfate to the supernatant, the other protein will also precipitate at a certain saturation rate. You can again centrifuge it, discard the supernatant and resolubilize the pellet.
Dialyze them against your buffer to remove excess amount of ammonium sulfate.
This method is non denaturing. You can also use this method if you want to concentrate your proteins.
Pankaj Parab
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