IPG ampholyte question

Hi!! I use IPGs for separation of proteins. But i can't understand one moment - if ampholyte have immobilized in IPG, why sample buffer for rehydratation contains ampholyte again?

The ampholytes are added to the buffer and IPG strip to stabilize the pH and act as carriers. They contain both a positive and negative charge (zwitterion).

In order for proper separation of proteins to occur during isolelectric focusing, a pH gradient is formed by  ampholytes (amphoteric buffers) having high buffer capacities at their isoelectric point (pI). The buffer outside the IPG strip must contain the carrier ampholytes because the gel needs to be in equillibrium with the buffer surrounding it through which the current is passing.  One can imagine leaving the ampholytes out of the buffer would cause the ampholytes to 'leach' out of the strip into the buffer to create equillibrium and thereby destroy any isoelectric focusing ability.

Rus

Thanx a lot!! I understand. Can i ask one more question? We have a dispute in our lab. Are ampholytes immobilize covalently in IPG or they just copolymerized with acrylamide and can move during IEF?

I am no chemist, but my understanding in the ampholytes move towards the charge poles exactly like the proteins, this is what actually forms the isoelectric focusing gradient. 

Rus

Hi tegrion and Rus

2DE has 2 era

the former one used IEF using carrier ampholytes

The major poblem during that was the cathode drift as the carrier ampholytes based pH ranges tend to drift towards carhode during the prolonged focussing times.

the current era uses IPG strips

where the covalently coupled immobilines are present that eliminated the problem of cathodic drift and resulted in the better resolution.

It was subsequently observed that a system using IPG strips and carrier ampholytes both provides the best reolution than either of the two used alone.

Finally

Carrier ampholytes are not immobilized covalently in IPG strips but the IMMOBILINES are ( two are very different) carrier ampholytes just add as additional buffering gradient and they drift towards cathode due to the same.

Hi Tegrion.

As Argerine and Rusty explained, IPG strips have better reproducibility and resolution due to a fixed gradient.  IPG strips are made using a mix of acrylamide derivatives or immobilines (covalently coupled  -COOH or amine groups to provide the appropriate charge). As I understand it is made just like a gradient gel using different combinations of immobilons to create diffrent pH gradients. I donot have the access to original papers, but may be you could find them in a library.

www.ncbi.nlm.nih.gov/pubmed/6643920

www.ncbi.nlm.nih.gov/pubmed/7142660

So, to answer your question, the charges moieties which make up the pH gradient in an IPG strips are covalently attached to the acrylamide and therefore immobile.  I think that adding ampholyte in your sample buffer enhances the gradient and improve resolution.  Both the ampholytes and your proteins would move to the cathode according to their charge as in a  regular IEF gradient.

Let us know how your 2D gels come out.

Hi again

I have papers from Angelika Gorg and Prof Righetti, the pioneers in the field. If any of you require these Let me know. I will be happy to email.

To add to Varsha's point

The two chambers for casting a gradient IPG has acidic and basic immobiline solutions respectively which is infact a mixture of zwitter ions (including the Aminoacids). The ratio and Pka range of the two type of solutions determine the pH range for IPG casting.

This has been explained in the manual by Angelika gorg entitled

Two-Dimensional Electrophoresis with Immobilized pH Gradients for Proteome Analysis

and the paper

Electrophoresis 1999, 20, 712- 717

Thanks to all for answers!! It's very helpful for understanding of method!

To Argerine - could i ask you to email the articles you have? do you remember my e-mail?

To all - Can i try to find out some details?

1. Recommended volume of rehydration buffer for 18 cm IPG 3-10 - 315 mkl. Is it critical to use specified in manual volume? Or we can use more or less volume of buffer for rehydration?

2. Recommended values for focusing for 18 cm IPG 3-10 - 10 000 V and 40-60 000 V-hr. What parameter is more important at finish - V or V-hr - what should be achieved at the end? Because sometime we have 60 000 V-hr and just 2 800 V.

3. Concentration of ampholyte in sample buffer. Recommended 0.4% for the IPG. When we tried 2.0% we got better resolution compare with 0.4%. BUT - voltage did not ramp up 10 000V, just 2 500 V. What should we do? Thank you!!

Its the total number of volt hours that are important and not the final voltage reached. Time could be a factor as increased focussing times can lead to the proteolysis.

Hi tegrion

You already have got the articles in the noon. Coming to yr queries

recommended volumes of rehydration buffer are there. The volumes are critical because under hydrated gel will pose problems in the migration of protein. sometimes your IPG strip can also burn and over hydrated gel will be having enhanced width particularly in the cathodic terminal.......... remember cathodic drift + electro endoosmosis.

It is the total Number of volt hrs that you have caried out yr focussing on is important irrespective of the total duration.

keep yr sample absolutely salt free, use lesser conc of ampholytes (not more than 0.8%) and you will reach the golden mark of 10000 Volts. you will see the current dropping with increase in VH. you will get perfect 0 micro ampere current if your gel will run perfectly. That is the point when yr proteins are absolutely focussed and carrier ampholytes have migrated to their designated pI.

If I remeber correctly You are wrking with plasma and If your sample is still the same I recommend you carry out focussig for 85000 VH which has worked well for me while using 17cm strip from Biorad.

Finally 2% carrier ampholytes are the upper highest limit for the carrier ampholytes....... I suspect that is the same reason yr final voltage is not reaching 10000V. Reduce it to 0.4 % or maximally keep it 0.8%. It will be sufficient for efficient focussing.

Hope these will wok out for you well

And finally a question from my side

How much protein are you loading on 18cm strip???

Thank you for yr ideas! Hi, Argerine! Yes, you are right, we work with plasma. But now we work with single protein separated from human plasma. We got a task to try separate different modifications if they are. So, load for 18 cm strip is 3-7 ug. At early stage of experiment we loaded up to 200 ug and got smearing. And now we hope to focus protein in spots. Some results are positive with 2.0% of ampholyte. Experiment with 0.4% ampholyte in progerss. Thanks for help!

Hi! What do you think about protein loading? Is it critical to load one protein (5 ug, for example) or protein mix (the same 5 ug total protein) for focusing? Can somebody tell me why do we need wicks? We have a hot dispute about wicks.

The electrode wicks are special whatman paper strips. Their purpose is to absorb the salt ions during focussing. using electrode wicks improves the focussing. It is also recommended to change the wicks if you are using gradual voltage RAMP.

wicks should not be wet but damp. Do not forget to blot the excessive water.

Hi Tegrion.

I typically run 11cm strips but do use the wicks. As Satinder suggested damp wicks absorb the ions from your sample. If your sample has a lot of salt you may want to use the wicks. I am not sure if it would be needed for the plasma sample. You may figure it out but running duplicate samples one with the wick and the other without it.

Hi Tegrion

As per my last information, Plasma has reportedly more than 16,892 proteins validated. If you are loading a protein mixture then you have to load more proteins for screening the one of your choice.

And Do use electrode wicks if you are using neat plasma as it has 150mM salt.

Ideally I use to TCA/Acetone ppt the same and then follow with 2DE.

Otherwise, also electrode wicks are recommended.