IEF gels require high voltage with steps changes at specified time points. The recommended run conditions are as follow: 1h 100 V, 1h 250 V, 30 min 500 V. I have never done before any IEF so could you tell me if I could make it just using a power supply up to 300V?
For the IEF technique the third step is essential to achieve narrow focusing of the proteins. At the end of the run, the charge of the proteins is too low so proteins need a high voltage to migrate. Thus achieving high resolution.
and coud I overcome it performing an O/N final step? Would it work??
Dear Masood,
The point is that we do have a Mini-PROTEAN® Tetra Cell coupled to a PowerPac™ Basic Power Supply (up to 300 V). In our lab we do not have any other equipments, that is the reason why I was asking about the possibility of an O/N incubation at a low Voltage!
Hi Xabier,
As Sebastián has correctly mentioned, high voltage is essential for the focussing. Infact, in the miniProtean System the initial runs at 100, 200, 400, 500 V - they are all prefocussing steps and the last step that is the run at 750V for 3.5 h is the actual focussing (IEF) step. Running SDS-PAGE for longer duration, sometimes over night, at low current/voltage can be done without much problem in protein resolution because separation is done on basis of molecular weight.
For, IEF the BIO-RAD PowerPac 3000 is the ideal one. If you see the present version for IEF using the IPG strips, the Protean IEF Cell has in-built Powerpac for precise voltage control, Infact, the proper focussing of proteins depends upon precise voltage control. For IPG run very high voltage like is used. So, I believe , the Basic Power Supply unit may not work.
Last word - since you do not have any other alternative, you can run multiple number of capillary gels for longer duration and see how it works.! Tell us, then, how it worked.
Good Luck.
Hi Bimal,
What do you really mean when you say "you can run multiple number of capillary gels for longer duration "?
I presume, you are carrying out first dimension run (IEF) in hand cast capillary tube gels and not using IPG strips!
If it is so, in one batch you can run 16 capillary gels. Load your sample proteins on 7/8 capillary gels for IEF and if you are running (say ) at 300V, run the IEF for different durations like 8/10/12 hrs; at 8hrs/10hrs/12 hrs interrupt the focussing and quickly collect one or two focussed gels for second dimension run (SDS-PAGE in mini-Protean). Look for two things; first, if there is 2-D resolution; second, if resolution is there, at what time it is optimal.
This is what I wanted to convey. This presumption is based on the point that you have a 300 V capacity Powerpac and you do not have a Protean IEF Cell (from Bio-Rad) or such other system for running IPG strips for the first dimension run.
Please feel free to discuss further, if it is not clear.
I presume, you are carrying out first dimension run (IEF) in hand cast capillary tube gels and not using IPG strips!
If it is so, in one batch you can run 16 capillary gels. Load your sample proteins on 7/8 capillary gels for IEF and if you are running (say ) at 300V, run the IEF for different durations like 8/10/12 hrs; at 8hrs/10hrs/12 hrs interrupt the focussing and quickly collect one or two focussed gels for second dimension run (SDS-PAGE in mini-Protean). Look for two things; first, if there is 2-D resolution; second, if resolution is there, at what time it is optimal.
This is what I wanted to convey. This presumption is based on the point that you have a 300 V capacity Powerpac and you do not have a Protean IEF Cell (from Bio-Rad) or such other system for running IPG strips for the first dimension run.
Please feel free to discuss further, if it is not clear.
Dear Bimal,
You are right about I only do have the Miniprotean system and a power supply of 300 V capacity...However, I just wanted to know the IP of my protein. I am planning to buy these gels (pH 3–10 Ready Gel® IEF Gel, 10 well, 30 µl - 161-1111, Bio-rad) and use them in the Miniprotean system. Woul it work?
Regards,
You can check the running specifications (mA/volt ) for running these IEF gels, from the supplier. Alternatively, for one or two spots analysis one can outsource the job. Good Luck.
Dear Xabier,
Did you try those precast gels in Mini Protean? If yes, can you tell me the clues for best practise?
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