Hi to all,
I'm trying to express and purify a protein. Once the lysate was obtained, I performed an ion exchange chromatography followed by a size exclusion.
In the chromatogram of the SEC, i have an intense sharp peak with a shoulder, I was able to collect and analyze them by SDS-PAGE.
I was expected to have two species, since I had two peaks in the chromatogram, but unfortunately, on the SDS-PAGE I have a population of Bands having different weights.
How is possible since the S.E. Chromatography separates species having different Molecular weight and size?
thank you
Hi Antonio,
I am a bit confused as to whether your problem has to do with contaminants or different oligomeric states of the protein of interest. Try to use this loading buffer and see whether you get rid of the upper bands:
2X urea sample buffer:
50mM Tris HCl pH 6.8
1.6% SDS,
7% glycerol
8M urea
4% β-mercaptoethanol
0.016% bromophenol blue
Some common solutions to aggregation are increasing\decreasing the salt or pH (depending on the pI and the nature of the interaction), adding glycerol or EtOH, adding some detergent (e.g. Tween20, NP40, TritonX...), increase 2BME or DTT. If you have a chaperone contamination iwould suggest an ATP wash during purification.
Good luck.
paolo
Hi Antonio,
From the size exclusion, you can see the two peaks, one minor (#3) and another major (#4) eluate. We also notice that the first is a shoulder of the second, major peak. If you extrapolate the peaks #3 and #4, the overlap is extensive and this may explain the overlap of proteins detected by the protein stain.The first peak has, as judged by SDS PAGE, a high molecular weight protein species around 95kDa that is seen to be not so prominant in the second fraction, #4.
These results suggests that the resolution of the SEC is not very helpful. You can slow the flow of the chromatography, collect more fractions so that the overlap of the Mr ~ 90-40kDa is more spread out.
You are using a very sensitive dye, silver stain. It is not a quantitative protein stain and you may be able to obtain more information by using another, Coomassie based stain, "Simply Blue" from Invitrogen.
Ion exchange chromatography may also be more helpful in resolving proteins that are originating from whole cell lysis steps..
Good luck, Hediye.
Hediye.
Have you tried running some MW standards on your SEC column? Perhaps the flow-rate is too high as Hediye suggested. Are you using gravity flow or FPLC? Without the MW standards, it is hard to tell whether this is a problem with the resolution of your column or instead reflects that these proteins are associating (aggregates or possibly a protein complex).
Agree with Daniel. This is basic problem of column. You should wash your column with 10% methanol than water for overnight and equilibrate minimum 2 hr. Before running sample run MW standard.
You don't specify what type of column or buffer you are using. The volume scale suggests it is one of GE's 10/300 columns. I agree with the comments above that you are looking at some kind of protein association. The first peak is probably at the void volume so what you see there is various aggregates. Even the second peak has many bands and is probably also aggregates. Try to include NaCl in the buffer to suppress ionic interactions and 20% glycerol to supress hydrophobic interactions. The second lane on the gel is enriched for a ~25 kDa protein - is this your target? Given the number of bands you see I would try to optimize the ion exchange step first and look at a second step before going to size exclustion. When there are aggregates in the picture I have had good results using mixed mode - particularly Capto Adhere.
GEC is not a high resolution technique. You likely need an intermediate purification step, e.g. hydrophobic interaction to get a purified product. We typically use IEX-HIC-GEC at a minimum for overexpressed proteins.
To maximize GEC performance, include at least 100 mm NaCl in the elution buffer, limit flow rate to the mfgr recommendations, and do not load with more than 2-4% of the column volume.
To really maximize SEC resolution you can go even lower on flowrate than the recommendations. On Superdex 200 PG columns we have seen that resolution keeps increasing down to 2 cm/h flow rates (we did not test any lower). But be prepared for some looong runs. And transfer to production will be a nightmare :-)
But I still think you need to solve the aggregation problem and get your protein more pure before the SEC step.
I agree fully with Bo; from the volumes on your chromatogram you are most likely using a column of the 10/300 geometry (superose ou superdex, impossible to say). Therefore the first peak is certainly the void volume whereas the second is at the begining of the fractionation range. In any case you are most likely dealing with agregated proteins. And this must be solved first. It would help to know the expected size of your protein and the complexity of the frations recovered from the IEX. But I suspect that an additional chromatography step is to be considered as suggested above. As far as aggegation is concerned, modifying the expression conditions is always my first move (reducing growth T°, decreasing inducer concentration...if it's bacterial expression, which you dont' say)
HI Antoni
This kind of results are possible, because afte cell lysis only one ion exchanger is used for purification, there will be a junk of proteins remained in your sample from 97KDa to 25KDa, it is difficult to purify directly by SEC, you may need to purify them further with HIC or some other techniues. I do not know your target protein Kda but after seeing the SDS PAGE you can try superdex 75. The sample volume must not be more than 5% of the total bed volume.
This is not uncommon when using a junk of proteins there may be a chance of retention at a time, the two peaks are explaining the same.
I am also suspecting your choosen matix is not able to separating the low molecualr weitht proteins.
Daniel also pointed some thing we need to think about.
All the best.
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