Author Topic: OK, still having trouble! Please help me :(  (Read 8680 times)

rachelhauser

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OK, still having trouble! Please help me :(
« on: March 15, 2011, 05:34:57 AM »
Hi everyone, I followed several suggestions from people on the board, however I still have problems in my IEF, the voltage is extremeley low and I´ll probably never reach the 50 kVh I need to focus my bile samples.

The samples are sonicated, delipidized, desalted, concentrated by lyophilization and centrifuged before IPG rehydration, according to an article about bile samples, I followed everything they did by the letter! Bile samples don´t have many cells, so I probably don´t need to use a lysis buffer, even though I also tried this and couldn´t ressuspend the dried sample in the appropriate volume of rehydration solution.

I even use a low 0.5 % IPG buffer as recommended by GE, I´ve seen people say that too high % of this may cause high condutivity and therefore low voltages...  what ELSE can I do to try and get a good IEF run???

ElkeKS

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Re: OK, still having trouble! Please help me :(
« Reply #1 on: March 15, 2011, 06:02:51 AM »
The thing that puzzles me the most about your experiences is that you say the voltage is not reached, but the current is also not at the maximum.
Are you sure that your IEF apparatus is okay? - Because if you have a sample that has high conductivity, then the current limit (typically 50mA/strip) is reached before the set voltage can be achieved. As the conductive material (e.g. salts) moves out of the strip into the paper wicks, the current goes down, and the machine increases the voltage either to the set value, or to the value at which once again the current limit is reached.
So, if you tell your IEF machine to do a step to 1000V, and it goes to less than 1000V while the current is below 50mA/strip, there is something wrong with the machine, not your sample.

Just a thought,
Elke

Sjouke Hoving

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Re: OK, still having trouble! Please help me :(
« Reply #2 on: March 15, 2011, 06:06:36 AM »
Hi,
As Elke suggested, a check of the powersupply might be needed.

Second, why not starting with an easy sample, either a cell line, or a bacterial sample and try to see if you get a good separation.
You will get some hand on experience on the procedures. Use the General Protocols for example (see sticky).

Good luck,

sjouke

rachelhauser

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Re: OK, still having trouble! Please help me :(
« Reply #3 on: March 15, 2011, 06:10:05 AM »
Hi,. the current is very near to the limit while the voltage stays low:

What it should be:

Current Limit: 50 uA/strip
Voltage in step 1: 200 v

What happens:
Current: 30-40 uA/strip
Voltage: 12 v

leo_metal

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Re: OK, still having trouble! Please help me :(
« Reply #4 on: March 15, 2011, 06:23:02 AM »
Hi Rachel

But...
As your sample is desalted, I believe that the low voltage could be happening because the low IPG%. IPGphor doesn't applying large voltages because your sample isn't offering resistance to it. So, maybe you should increase the IPG% to force IPGphor to apply highest voltages. Actually GE recommends to use 0.5-2% IPG.
I used to apply 1.6-2% in my samples and my IEFs was OK.

See ya

ElkeKS

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Re: OK, still having trouble! Please help me :(
« Reply #5 on: March 15, 2011, 07:39:44 AM »
I don't agree with Leo. Assuming there is an electrical connection established between the electrodes (strip evenly rehydrated, damp paper wick making contact to gel and electrode, electrodes not corroded), then the machine should apply whatever voltage is programmed, UNLESS the current limit is reached.
So I think there is something wrong with the IPGphor.
Elke

rachelhauser

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Re: OK, still having trouble! Please help me :(
« Reply #6 on: March 15, 2011, 07:55:24 AM »
Hi guys (In defeated and sad tone of voice... )

Everything you said Elke has been done, the strip is evenly rehydrated, the paper wicks damp and in contact with gel, the electrodes perfectly placed and pratically new (the whole equipment is only about 2-3 years old and I´m the only one who´s ever used it)...

I´ve let the stupid thing run anyways, it´s now at a setpoint of 1500 V and 50 uA/strip, and has only reached 133 V and 40 uA... So it seems it´s really the IPGphor.

I´m going to take a trip to another lab (far away but it´s the only one), run a sample there and see what happens. If the sample runs OK there, perfect, I´ll have to contact GE and ask for a technical visit for my IPGphor. If not... (menacing drum rolls for when I talk to my advisor...)

I really appreciate all your help and suggestions, if I have any news I´ll update this thread. Thank you again all so much for your help.

rachelhauser

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Re: OK, still having trouble! Please help me :(
« Reply #7 on: March 15, 2011, 11:32:30 AM »
Ummm... after 6 hours of running at low voltages the thing just upped its voltage to the right setpoints and now seems to be following the right settings... WHAT ON EARTH HAPPENED? After 6 hours it can´t just be salts in the sample, can it?? Maybe the IPGphor is really showing some problem that makes it take forever to get to the right voltages??

dfried

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Re: OK, still having trouble! Please help me :(
« Reply #8 on: March 15, 2011, 10:53:05 PM »
Rachel,
Following this thread, it really does seem more like your IPG focusing unit is the problem.  Just because the unit is only 2-3 years old doesn't mean that it cannot be malfunctioning. If you cannot test the system with an easy sample, just try focusing a rehydrated  strip with no sample in it. (And for these units, I also would not recommend more than 0.5% IPG buffers).

David

rachelhauser

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Re: OK, still having trouble! Please help me :(
« Reply #9 on: March 16, 2011, 04:01:51 AM »
I tend to agree David,

I called GE Brazil yesterday and left a message and contact number, however they haven't called back... just left them a second message right now, saying I need urgent technical assistance.

Meanwhile I'll run the IPG strip that got to OK voltages yesterday and see what happens... it got to the right number of kVhs even though it took forever, so I may be able to get some results.

Thank you all! :)

rachelhauser

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Re: OK, still having trouble! Please help me :(
« Reply #10 on: March 18, 2011, 07:08:33 AM »
Hi guys, I just tested a rehydrated 18 cm pH 3-10 strip without sample (standard GE solution with 0.5% IPG buffer), and tested different voltage setpoints, and got the following behaviour:

- 200 V setpoint and 50 uA/strip --> The voltage went up immediately to 200 V and the current was 23 uA

- 300 V setpoint and 50 uA/strip --> The voltage went immeadiately up to 300 V and the current was 35 uA

- 500 V setpoint and 50 uA/strip --> The voltage went up to 450 V, stayed there for a few seconds, went down to 440-435 V and the current was 52 uA and the A was blinking.

- 1000 V setpoint and 50 uA/strip --> The voltage went up to 430 V and the current was 52 uA and the A was blinking.

This does seem to indicate a problem with the power supply, right?


Sjouke Hoving

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Re: OK, still having trouble! Please help me :(
« Reply #11 on: March 18, 2011, 07:18:55 AM »
Hi,

I would say so that the problem is with the instrument. It can happen sometimes that the max voltage is not reached due to salts in the sample (high current). These salts migrate out of the strip (and absorbed by the paperwick at the electrode). As a consequence the current drops and voltage will increase again. Unless you are using seawater in your strip it doesn't look like the problem in your case...

It's time to get a technical support from GE.

sjouke

rachelhauser

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Re: OK, still having trouble! Please help me :(
« Reply #12 on: March 18, 2011, 09:33:39 AM »
YAY! :D

(At least it´s an instrument issue and not my sample or any stupidity on my part... yet...!)

Thank you Sjouke, as always you´re extremely helpful!

I´m now waiting for GE to call back, probably only on Monday though, to set up the date ASAP for technical assistance :)

dfried

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Re: OK, still having trouble! Please help me :(
« Reply #13 on: March 18, 2011, 02:35:48 PM »
I agree it is likely the unit.  But just to double-check, you are using new/fresh urea with your solutions, right?  I'm wondering whether if using old/heated urea if the break-down to isocyanate would increase the current significantly (in addition to carbamylating the lysines and shifting protein pIs ...).  Just a thought.

Hope all goes well with the service visit.

David

rachelhauser

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Re: OK, still having trouble! Please help me :(
« Reply #14 on: March 18, 2011, 03:50:34 PM »
Hi David,

I prepared two strips, one with a rehydration solution that was frozen in one-time use aliquots at -20 a few months back, and one with a fresh solution, and both gave me about the same results! Better safe than sorry, if I hadn't done that I'd probably be wondering about the use of an "old" solution too :)

GE hasn't called me back yet, so I'll wait anxiously for Monday to arrive!