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Re-using solutions, protein quantification assay, lyophilization and IEF problem


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#1 rachelhauser

rachelhauser

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Posted 11 March 2011 - 03:49 PM

Hello everyone, I have a few quick questions about 2D solutions:

1 - Does anyone re-use the dry strip cover fluid (mineral oil that covers the IPG strips)?

2 - Does it matter if some of the oil drips onto the IPGphor surface (inside, where the Manifold is put), either on the golden surface or the black one, and then you run the IEF?

And about sample quantification:

1 - Which is better, 2D quant kit, Bradford or Lowry? (I only have these 3 available). I ran a Lowry and 2D quant and got a value of 40 ug/mL in my sample, so I concentrated my sample by lyophilization (don't have access to a SpeedVac), and ressuspended directly in 250 uL standard rehydration buffer for 13 cm IPG strip rehydration. However, it seems that the sample got too concentrated, my strip was very dark, the colour of the sample (green, it's bile) and the voltage on my IEF was extremely low, so I saw nothing on my gels, so I think I should re-do the quantification procedure...

Important: I know about salts and how they should be avoided, so the first thing I do after I collect my bile samples is desalt using G-25 Midi Trap columns by GE Healthcare. I assume they work, so my sample is desalted before lyophilization, so I SHOULD be getting good IEF and gel results. That's why the only thing I can think of is the protein quantification that is going wrong somewhere.

Any help would be very appreciated!

Details:

Maniold IEF on IPGphor 3
13 cm 3-10 IPG strips
Slow IEF, starting at low voltage, going up to 8000 v gradually over about 15 hours.
Desalted bile samples (5 kDa Cutoff, should get rid of most interferents).
Rehydration buffer: standard GE recipe from the handbook, Urea, CHAPS, DTT added just prior to use, IPG Buffer.

=)

Edited by rachelhauser, 11 March 2011 - 03:52 PM.

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