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Polyacrylamide gel running

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Polyacrylamide gel running

Postby Akshay » Mon Jul 02, 2007 4:32 pm

Hi, I am new to running polyacrylamide gels (used agarose gels in our previous Experimental modules)...when i run a 12% 1mm gel, it takes a really long time (>4 hours) for the marker line to reach the base of the gel (at 90V), even though the protocol mentions 2.5 hours. Does anybody have any reasons why?
Also, the tank I am using for gel electrophoresis has space for 2 gels. I noticed that the marker line in one of the gels is always faster by about 2cm than in the other gel. I thought both should run at the same rate?

Any solutions would be really helpful,
Thanx
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Postby blcr11 » Tue Jul 03, 2007 7:42 pm

Check that the buffers are made properly. If your buffers are inadvertantly at say half-strength, that might explain the slow running. Both sides of the gel unit should run the same provided the gels are of the same composition and thickness.
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Re: Polyacrylamide gel running

Postby Ewa » Wed Jul 04, 2007 6:07 pm

hi

you may try the following

- check again concentrations of acrylamide stock, TEMED and APS you use to prepare your gels - 4.5 h is typical for 16% protein gels (5 cm run), are you sure everything is ok with your 'ingredients'?

- use freshly prepared gels, do not store them, and if you do - keep the moisture by wrapping them with wet paper towel etc. do not let them dry!

- go up to 100 V for tricine gels, up to 150 V for glycine (Laemmli) gels - I assume you run protein gels, right?

- check pH values for gel buffers and running buffer(s)

Akshay wrote:Also, the tank I am using for gel electrophoresis has space for 2 gels. I noticed that the marker line in one of the gels is always faster by about 2cm than in the other gel. I thought both should run at the same rate?


- check the platinum wires (the electrode - if you are able to see it) and the current (you should be able to see air bubbles running in the buffer)

hope any of this helps :)

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Postby Akshay » Thu Jul 05, 2007 11:59 am

I am pretty sure the ingredients I used are of the right concentrations for a 12% PAGE, and I freshly prepare the ge. I have a feeling maybe it runs for so long is because we are re-using the running buffers in this lab - maybe the buffer is depleted or something.

'go up to 100 V for tricine gels, up to 150 V for glycine (Laemmli) gels'
I am using 1X tris-glycine buffer for the running buffer. My supervisor says to use 90V for the electrophoresis, but I read much higher voltages in some of the protocols, and as Ewa mentioned. Could someone please inform on what would be the max. 'safe' voltage that I can use? Also, is there some correlation between voltage used and the clarity of the bands?
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Postby cdcruz » Thu Jul 05, 2007 4:43 pm

increased voltage lowers the run time for the gels, but also lowers the resolution of the bands. conversely, a low voltage lengthens the run time of the gel but provides crisp, clear bands. it is a vicious game of give and take, when short on time and patience. 100V should allow for a run time of ~3 hrs and produce fine bands.

your supervisor seems to have reason to suggest 90V, though, and may be desiring a more crisp resolution. will there be any western blots following this electrophoresis? if so, clarity is key. despite the longevity of the run time, i would proceed w/ 90V.
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Postby andyde » Thu Dec 06, 2007 1:43 pm

Akshay wrote:I am pretty sure the ingredients I used are of the right concentrations for a 12% PAGE, and I freshly prepare the ge. I have a feeling maybe it runs for so long is because we are re-using the running buffers in this lab - maybe the buffer is depleted or something.

'go up to 100 V for tricine gels, up to 150 V for glycine (Laemmli) gels'
I am using 1X tris-glycine buffer for the running buffer. My supervisor says to use 90V for the electrophoresis, but I read much higher voltages in some of the protocols, and as Ewa mentioned. Could someone please inform on what would be the max. 'safe' voltage that I can use? Also, is there some correlation between voltage used and the clarity of the bands?


Didn't anybody explain you the principle of discontinuous SDS-PAA-Gel electrophoresis?
1)The sharpening of bands is a result of the drop of current at the moving border if the slow glycinate ion (in the buffer) and the fast chloride ion (in the gel). If Chloride ions are present in the buffer this effect is diminished.
2)The buffer is electrolysed at cathode and anode (remebmber the bubbles at the electrodes?) which changes the pH of the buffer.
So never reuse the buffer in SDS-gel electrophoresis. If you don't trust your buffer ( or the one who prepares it) buy readymade stock (available from MicroMol GmbH and other suppliers).
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