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power supply electrophoresis - (Mar/21/2011 )

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Hi,
I need your help to understand my problem, I run 2 gels in parralel at constant current, 40mA for the stucking, (since I was used to run at 20mA for one gel), and the double when the proteins reach the resolving gel, ( for one gel, I run at 40mA). so, in this case of 2 gels, I used like 70 to 80 mA, constant current, I have to increase the voltage to reach 80mA, I go up tillaround 400v. then, I run, after about 30min, I realised that my current isnt constant and decreased to 33mA instead of 80., also the voltage decreased. so , I stopped the runinng and restarted again, I saw that isnt possible to reach the desired current, since the voltage button was blicking! then, I found that the cable of the cup passing the current to power supply was brocken, so, I used another cup, when I run, the power supply gives error allarm and couldnt work! so, I decided to change the power supply and use one of biorad, and I could ran with constant voltage, 100V, it was fine!

My question is whether the power supply could break because of reaching high voltage 400V or more!!, since Its written on the instrument powe supply 400V-400mA electrophoresis.
also, do you thing that something in my buffer or gels that could disturb the current and consequently the running?
thanks

-luciana-

During electrophoresis the electrolytes in your buffers get depleted (you can observe this as bubbles forming on the wires)... this is why the current and voltage drop. You should not need to run the gels at over 400 V unless you are running very long gels (30 cm long). For most mini gel setups (10 cm or less) you should only need to run at 120 V.
I suspect you have also got it a bit wrong on the multiplying things out, you should only need to run the gels at 20 mA, as the powerpack should adjust for each output, unless you are running the gels in a series circuit (if you can recall the basic circuits you did at school).

-bob1-

bob1 on Mon Mar 21 22:17:54 2011 said:


During electrophoresis the electrolytes in your buffers get depleted (you can observe this as bubbles forming on the wires)... this is why the current and voltage drop. You should not need to run the gels at over 400 V unless you are running very long gels (30 cm long). For most mini gel setups (10 cm or less) you should only need to run at 120 V.
I suspect you have also got it a bit wrong on the multiplying things out, you should only need to run the gels at 20 mA, as the powerpack should adjust for each output, unless you are running the gels in a series circuit (if you can recall the basic circuits you did at school).



You are right, thanks, I saw bubbles, but I thought its the running that started. Im not expert in the field.
I was running 2 gels in the same apparatus, only one circuit.
I was not used to run at constant current, but at constant voltage! I know now the mistake. What is better according to use, constant current or voltage? if u set a constant current, do u set a voltage or u dont ?
another thing, do u check one of the parameters current or voltage if u choose constant one? or u just set one, and run??
im sorry, for these stupid questions.

-luciana-

luciana on Mon Mar 21 23:04:54 2011 said:


bob1 on Mon Mar 21 22:17:54 2011 said:


During electrophoresis the electrolytes in your buffers get depleted (you can observe this as bubbles forming on the wires)... this is why the current and voltage drop. You should not need to run the gels at over 400 V unless you are running very long gels (30 cm long). For most mini gel setups (10 cm or less) you should only need to run at 120 V.
I suspect you have also got it a bit wrong on the multiplying things out, you should only need to run the gels at 20 mA, as the powerpack should adjust for each output, unless you are running the gels in a series circuit (if you can recall the basic circuits you did at school).



You are right, thanks, I saw bubbles, but I thought its the running that started. Im not expert in the field.
I was running 2 gels in the same apparatus, only one circuit.
I was not used to run at constant current, but at constant voltage! I know now the mistake. What is better according to use, constant current or voltage? if u set a constant current, do u set a voltage or u dont ?
another thing, do u check one of the parameters current or voltage if u choose constant one? or u just set one, and run??
im sorry, for these stupid questions.

bob1 got that a little wrong, when running two gels in parallel you can either use the same voltage as one gel or twice the current. the current splits between the gels, equally in a perfect world but the world isn't perfect so you have to keep an eye on the gels and they may not finish together. the voltage is across the entire system.

which to use as constant depends on your preference and the recommendation of the apparatus' manufacturer.

whichever you choose to be constant you will still want to set the other as a limit so that you don't over stress your system.

and, the bubbles on the electrode wires are due to the electrolysis of water.

-mdfenko-

mdfenko on Tue Mar 22 16:27:51 2011 said:


luciana on Mon Mar 21 23:04:54 2011 said:


bob1 on Mon Mar 21 22:17:54 2011 said:


During electrophoresis the electrolytes in your buffers get depleted (you can observe this as bubbles forming on the wires)... this is why the current and voltage drop. You should not need to run the gels at over 400 V unless you are running very long gels (30 cm long). For most mini gel setups (10 cm or less) you should only need to run at 120 V.
I suspect you have also got it a bit wrong on the multiplying things out, you should only need to run the gels at 20 mA, as the powerpack should adjust for each output, unless you are running the gels in a series circuit (if you can recall the basic circuits you did at school).



You are right, thanks, I saw bubbles, but I thought its the running that started. Im not expert in the field.
I was running 2 gels in the same apparatus, only one circuit.
I was not used to run at constant current, but at constant voltage! I know now the mistake. What is better according to use, constant current or voltage? if u set a constant current, do u set a voltage or u dont ?
another thing, do u check one of the parameters current or voltage if u choose constant one? or u just set one, and run??
im sorry, for these stupid questions.

bob1 got that a little wrong, when running two gels in parallel you can either use the same voltage as one gel or twice the current. the current splits between the gels, equally in a perfect world but the world isn't perfect so you have to keep an eye on the gels and they may not finish together. the voltage is across the entire system.

which to use as constant depends on your preference and the recommendation of the apparatus' manufacturer.

whichever you choose to be constant you will still want to set the other as a limit so that you don't over stress your system.

and, the bubbles on the electrode wires are due to the electrolysis of water.


ok! thanks for your intervention!, Do you think the method I use of doubling the current and limiting the voltage is right?
the problem, is that the power supply works at the biginning, but during the running when the proteins get a bit down in the resolving gel, I saw unstable values either on the current or the voltage! what could be the problem? is it the resistance that was very high inside the chamberof electrophoresis?..
also, I stopped this running and decide to run at constant voltage of 100V, I see that the current is 10mA, is it normal according to you?
Im willing to run again 2 gels in parallel, Im afraid to have the same problem! I would run at 100V for 2 gels, without lmiting any current. do you think it could be fine??

-luciana-

a rapid drop in current would indicate that the electrode buffer is weak. make sure that it was prepared properly (concentrate and dilution).


how did the gels look when you finished?

-mdfenko-

mdfenko on Tue Mar 22 20:54:04 2011 said:


a rapid drop in current would indicate that the electrode buffer is weak. make sure that it was prepared properly (concentrate and dilution).


how did the gels look when you finished?


Hi mdfenko, the running buffer is Tris-Glycine SDS: trizma base+GLycine+SDS 0.1%. I have the precise concentration in my notebook in the lab!
It was prepared 5 days before the day of the experiment and stored at 4C. (ALready used for 1 gel electrophoresis I made last week that was fine, and restored).
In fact, I did coomassie staining on those 2gels run in parrallel, I saw after overnight destaining, that the gels looks a bit clean, with some blue spots in the center, that according to me due to the coomassie staining, but there are no proteins on those 2gels!!
another strange thing, is that I made the transfer of those gels in filters, I did a mistake by making in contact the white side (positive charge)of my sandwich with the black (negative) of the tank chamber for wet transfert, but the other sandwich was white side against white side of the tank, but after ponceau, there are no proteins!!! so, its really strange, either no transfert because of converting the position of sandwich, either the transfer was done but not on my filters but on the wathman paper or the sandwich support...?? Its really strange experiment since the runinng!!
Thanks alot for your help

-luciana-

Buffers do deplete with each run, so re-using tris-glycine buffers is not usually a good idea.

-bob1-

and storing buffers with sds at 4C is not advised. the sds will crystallize at low temperatures then you have to ensure that it is completely redissolved prior to use.

-mdfenko-

mdfenko on Wed Mar 23 14:50:01 2011 said:


and storing buffers with sds at 4C is not advised. the sds will crystallize at low temperatures then you have to ensure that it is completely redissolved prior to use.


Hi,
I prepared Tris-Glycince-0.1%SDS 10X, and dilute to 1X, and I put at room temperature, I have to run the 2 gels tomorrow, hope this buffer will be ok! Also, I chose one gel 10% SDS-PAge and the other 12%gel. I will run together!

-luciana-
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