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I can't resolve 2 proteins (LC3-I and II) in Tricine gels


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#1 autophagator

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Posted 28 December 2009 - 01:16 PM

I am trying to run a 16,5% Tris-Tricine gel with glicerol, in search of two proteins: LC3I (14KD) and LC3II(16kD), autophagosomal proteins. I am running the gel using separate Anode and Cathode buffers and I run it 80V and then turning up to 100V for 5 hours. Also, I use the Tricine sample buffer. But, when I check my gel(revealed with horseradish-peroxidase and the chemiluminescence detection system) only appears one bandˇˇ
So, the time of run it´s not enough?
Please help me out.Thanks in advanceˇ

#2 Prep!

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Posted 28 December 2009 - 07:54 PM

Correct me if i am wrong.. but i thought we can resolve a difference of 2 kDa in gradient Gels easily!!! in the conventional lamelli system itself!! - may be a gradient from 16-20% or so??!!!
We can comment on the time of run only after seeing the gel auto!! is it in the middle of the gel or top or bottom etc??!!!
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#3 mdfenko

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Posted 29 December 2009 - 09:12 AM

are you running a minigel?

how far does the bromphenol blue migrate?

for 14-16kDa you don't need a 16.5% tricine gel. 10% with a 10% spacer gel (if your gel is big enough, not a minigel) should work fine to separate them.

you can also run a gradient with tricine (10-16.5 or 20%) but the bands may get closer than with a non-gradient.

if you choose laemmli, as pi suggests then 15% should be fine (10-15 or 20% if you choose to run a gradient).
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#4 autophagator

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Posted 04 January 2010 - 07:24 PM

Yes...I can´t, but my gels are discontinous, not in continuous gradient: 4%(stacking)+12%(spacer) and 15% (resolving) and in a mini gel of 10cm. The front dye reaches the bottom of the gel (and beyond..) and I keep seeing one band, when I should see two bandsˇˇˇ I dont know what to do.May be I must increase the voltage of the run?Do you preset the current and voltage simultaneously before run a gel?or the current is seted in 400 mA always?
Well, anyway.. thanks for your time, I will continue trying...

#5 autophagator

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Posted 04 January 2010 - 07:40 PM

Yes "md", its a minigel, and the front dye reaches the end of the gel...Recently, I tried with a 12%(spacer)+15%(resolving)gels, with Tricine system in a minigel, but I could not separate it.
Now I think that the voltage is to low, since in the bibliographical reference of tricine system, says that the current must be seted in 80mA AND the voltage in 30+100V...
Well, I thinking change to Laemmli system if this problems persist..
Anyway, thanks a lot "md".

#6 mdfenko

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Posted 05 January 2010 - 11:06 AM

Yes "md", its a minigel, and the front dye reaches the end of the gel...Recently, I tried with a 12%(spacer)+15%(resolving)gels, with Tricine system in a minigel, but I could not separate it.
Now I think that the voltage is to low, since in the bibliographical reference of tricine system, says that the current must be seted in 80mA AND the voltage in 30+100V...
Well, I thinking change to Laemmli system if this problems persist..
Anyway, thanks a lot "md".

i think you should try a gel with 4% stack, 10% spacer and 10% running gel (running gel contains glycerol, spacer does not). 15-16% may be too high for your light chains to migrate well.

run at 45-50V for 15 minutes then 110V for 1.5-2 hours (until tracking dye reaches the bottom of the gel).
talent does what it can
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