First, you should use gels for determining pIs rather than the Rotofor.  The Rotofor is not outdated.  It is a preparative instrument, not an analytical one.  The pH measured in each fraction is an average of the ampholytes captured there.  Those will change depending on the physical positioning of the insert into the chamber so you might think the runs are not reproducible.  They are reproducible enough for prep work.   If gels don't work, something else is going on.

Second, the best way to use a Rotofor is to make two runs with the same sample.  Collect the fractions containing the protein of interest and add water or dilute buffer to the 20 ml chamber volume.  Do not - repeat, do not - add more amphollytes regardless of what is said in the instruction manual.  Your collected fractions will contain enough ampholyte for focusing and the pH range will be nearly centered on the pI of your protein.

Also, too much buffer will give high initial currents. The buffer ions will eventually clear if you wait long enough and the gradient will form just fine.

So, please use gels for determining pIs and the Rotofor for preparing your protein.

Hi Mustafa. I am amazed that Rotofors are still in use.

I used Rotofor in my post-doc and the main problems I found is that

1) glycosylated proteins like transferrin never give a single precip. band because different glycosylations result in different pI. I preferred to use serum albumin as an indicator because it is non-glycosylated and there is a lot of it in media and blood.

2) if you have more than 0.1M buffer in your sample, it will not focus on the Rotofer.  Ampholines are just a mixture of buffer molecules with different pI's that focus proteins based on their pI. Any added buffer will screw up the gradient.

First of all rotofers are outdated because of these types of problems, which hinders to give reproducible results. I can only suggest that you try to use different ratios of water to ampholites (it will be still very time consuming).

Thank you for your interests.  As both of you said, it is an old labrotary instrument, but I am working on antibodies and I need to learn their isoelectronic points for better elution. My lab's rotofor was not being used for a couple of years, for pI detection it looked the best option. Our lab also has a 2d electrphoresis system but i thought that it was harder to understand. If you have a better and easier advice it would be a pleasure to learn. 

So wonderful, if only you or we knew the isoelectric point of your proteins.......HOW SIMPLE THE WORK WOULD HAVE BEEN. You need not have used the rotofers. That is why I have asked you try various permutation and combinationd of water and ampholites to get to the answer.

Hi Mustafa. I applaud your tenacity!

However, Rotofor was designed for preparative isolation of proteins according to pI. We are talking about focusing grams of proteins in a single run.

If you just need to know the pI of antibodies, then you can just do the IF part of the 2D. 

Dear Mustafa

Many expriments have demonstrated that Rotofor present more difficult to monitor PI. It will be hard if you decid to approach by ratio water/ampholites

Thank

Marius

First, you should use gels for determining pIs rather than the Rotofor.  The Rotofor is not outdated.  It is a preparative instrument, not an analytical one.  The pH measured in each fraction is an average of the ampholytes captured there.  Those will change depending on the physical positioning of the insert into the chamber so you might think the runs are not reproducible.  They are reproducible enough for prep work.   If gels don't work, something else is going on.

Second, the best way to use a Rotofor is to make two runs with the same sample.  Collect the fractions containing the protein of interest and add water or dilute buffer to the 20 ml chamber volume.  Do not - repeat, do not - add more amphollytes regardless of what is said in the instruction manual.  Your collected fractions will contain enough ampholyte for focusing and the pH range will be nearly centered on the pI of your protein.

Also, too much buffer will give high initial currents. The buffer ions will eventually clear if you wait long enough and the gradient will form just fine.

So, please use gels for determining pIs and the Rotofor for preparing your protein.

Mustafa, I agree with everyone, and it's easier to run IEF gels than use the rotofor, and you can just stain them with Coomassie. A protein like transferin that has many posttransltional modifications and is Fe-binding will undoubtedly give multiple pIs and will streak if not properly denatured. I used to desalt my samples with spin columns and dry them in a speed-vac before resuspending is IEF sample buffer and it works well.