I just want to know what are the parameters are that we have to set in an IEF cell for the focussing of a 7cm IPG strip. Initially we followed the guidelines as provided in the instruction manual (0-4000V, Rapid,8000Volt Hours at 20 degrees). I am just wondering whether I can follow a three-step protocol instead of only one step. The three steps that I have set in the IEF cell are:
S1: 250 V for 15 minutes
S2: 4000 V for 30 minutes, rapid at 20 degrees
S3: 8000 V-Hr.
S4: Hold on at 500V
Can I set these as the default parameters for running a 7cm IPG strip (pH 3-10).
Please provide me with suggestions.
The most important variable to be reached in an IPG strip focusing run is the total Volts/hour. Because this depends on the composition of each sample, it is best to set your steps to a final Volts/hour achieved, rather than x volts in y minutes.
For 7cm strips loaded with 50-100ug of total protein, a total of 6,500 volts/hour is enough, 50uA constant current.
I have used in the past the following program for Arabidopsis suspension cell culture soluble proteins in Urea buffer:
1-100V until 5 V/hour reached
2-3500V until 6500 V/hour reached
For larger (24 cm) strips a more complex program is needed.
I have used 13 cm strip (pH 3-10)and I have followed the parameters of 11cm strip (because it was the close length to my strip) to set in an IEF cell , which I followed just 3 steps and ignored the 4th step . Exactly as the guideline in the instruction Manual said (follow the instruction for 11 cm strip).It worked perfectly with me.
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