I am trying a IEF with precast Biorad gel, anode buffer H3PO4 7 mM and cathode buffer NaOH 100 mM. I am not able to get the stepwise power conditions that the manufacturer recommends. It means it is impossible to set 100 V constant 60 min + 250 V constant 60 min + 500 V constant 30 min, keeping starting and final current 5-25 mA/gel.
Has anyone had to deal with this kind of problem? How to solve it?
Dear Sandra
It is important to increase the voltage step by step (to prevent damage to the proteins and gel) and reach the maximum Vhr (for finding the best focusing).
Biorad also provide IEF strips that work better and produce good results. I work with them for long time and published several papers. These strips have different size and pH range and working with them is simple and easy.
If you have any questions, please don't hesitate to contact me
With best regards
Dear Sandra,
Is the problem with the resistance of your system? - If you set the voltage to 100 V, and carry out the electrophoresis for 1 hr, how the power changes?
The conditions depend on the temperature as well. Try to chill your system, if it is possible. For example: place the gel and buffers to a fridge before electrophoresis (or during electrophoresis). It may solve your problem.
You can also contact the local bio-rad office for assistance.
Good luck! :-)
Best regards,
Gizella
In the early phase of the experiment you might be run the process in power mode. So, set the power supply at 10 W for instance. In this case the electrophoresis starts at sufficiently low current and voltage, thus keeping the condition 5-25 mA/gel during the process.
To start with, i do IEF with Protean i12 IEF Cell. What is the instrument model you are using? Default programs are preloaded in IEF i12 Cell. Two options are always present. The rapid ramp protocol or the gradual ramp protocol.
If you have cleaned your protein sample with Bio-rad cleaup kit before resuspension in Readyhydration buffer, i suggest using i12 IEF rapid ramp protocol can do a pretty good job with bio-rad IPG strips.
Please specify your instrument
i have the same problem but i read in some papers that voltages so high than 12000 v must be used if not reach such system use 500 v for overnight. my problem is the current as my current reach 0 mA after 5 to 10 minutes of running in 200 v.
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