The protein (55KDa) has low contentration. It is purified via ammonium sulphate salting out and desalted with pD10 column. Will working with SDS-PAGE (10% resolving and 5% stacking), a drag is observed. How can we remove it?
You have too much ammonium sulfate in your sample. The high ionic strength distorts the electrophoresis.
What is the buffer composition for the desalting PD10? How much volume do you apply to the PD10?
I think that this might happen due to high voltage or overheating.
It is a while ago that I run my last gel, but if I remember it correctly, with high voltage or overheating you would not have smearing, but not these wide bands.
I would agree with Achim Recktenwald that most likely you have excess salt or ammonium sulfate in your sample. Since the MW markers look good except where distorted by the adjacent lane, the problem is probably not with the gel but with the samples.
Hi,
I do agree. It only seems you did not get rid of the salts you added to purify your protein ... So, you could probably pass it through one or twice more on your collumns. Check it out your buffers salts preparation, just to be sure they're ok. Right?
Wi sh you al the best.
Dragging could be due to high salt as others have pointed out. But you have desalted the sample in pD10 column. I do not see much smear in the first well; samples are looking okay. If the desalting is not efficient, you could dialyze the sample and reload and check. Smear could be due to protein degradation also. Use a protease inhibitor or a cocktail during the extraction and try again.
I have experienced this problem two times before ,the problem was in the gel quality and voltage distribution of the Electrophoresis device,I advised you to make a new gel and increase the percentage of the resolving gel to 12% and change the Electrophoresis device
Achim Recktenwald my protein is Laccase, we have tried pD10 desalting with HCl-KCl buffer and Cirtrate buffer pH:3. Ammonium sulphate fractionation is 30-90%
Achim Recktenwald Arsh Shrikant Chavan we are maintaining 70V for stacking and 110V for resolving which I think should not cause dragging. Also wide bands are because we have merged 2 wells in the gel so as to load more protein as our protein conc. is low.
Ridhima Wadhwa, I still think it is too much salt in your sample.
You could try one thing I did many years ago with a similar problem running too salty samples on a Pharmacia PhastSystem.
At the start of your electrophoresis, when everything is still in the stacking gel, run your gel at a low voltage for a while. You will have to experiment with the time; I would try 5 - 10min. With the low voltage only small ions will move, the proteins stay where they are. This will desalt your samples to some degree. Then switch to the normal voltage to run your gel.
In my case it worked like a charm, but I never tried it with normal electrophoresis gels, just these time Phast gels.
Achim Recktenwald thank you so much for your suggestion. I shall try your suggestions. Could you please suggest how should I get a thick prominent band of my protein?
You will have to load more material.
As I assume you loaded as much volume as was possible, you have to concentrate your sample. Check for a spin filter with the lowest molecular weight cut off you can find; 1000 to 3000Da should be fine, as anything smaller than 4kDa are peptides, which I assume you are not interested in.
Membranes tend to bind protein, especially when you force the protein into the membrane with a high g-force. Some proteins bind more then others.
If this is a problem with your protein, try something I did many years ago. I put some circular filter paper into a petri dish. I then put a membrane filter of low cut off onto the filter paper. And on top of the membrane I put a few drops of my sample. The water from the sample would be sucked slowly through the membrane into the filter paper.
I remember that the problem was to find the right membrane filter; it had to be slightly hydrophobic to maintain a droplet that did not just spread on its surface, but not too hydrophobic so that water could penetrate.
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