problem blue native

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albertopz
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problem blue native

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We are currently performing a Blue Native gel electrophoresis to visualize intact mitochondrial complexes from mice heart following the method published by Schagger´s group (Nature Protocols  1(1): 418-428, 2006). We have tried to do it in both mitochondrial crude fraction and mitochondrial enriched fraction using Dodecylmaltoside as detergent and Comassie Blue G-250. For the purpose, we are using PhastSystem Separation-Control and Development Units 220 VAC (18-1018-24), PhastGel Gradient – 4-15 (17-0678-01) and PhastGel Buffer Strips – Native (17-0517-01) from GE. The method that we are using is the pre-saved method 1 called “Native” in the PhastSystem Separation Unit. However, we are experimenting a big problem: when the sample is running under step 3 (around 70-75 AVh) a drop in the  mA is experimented (from 10 mA to 0.4 mA; 400 V and 0 W). With this level of mA the electrophoresis is almost stopped on half-2/3 of the gel.  I don´t understand the reason of this problem. Do you have any idea?

Thanks

qiang wang
qiang wang's picture
4-15% gradient for native gel

4-15% gradient for native gel is too much, I won't go above 10%

for the extremly low current, it is fine, it takes hours to ovenight to run a BN gel. In your case of 4-15% gradient, I bet you won't have any protein complexes at the lower half of gel.

albertopz
albertopz's picture
But the Shagger protocols,

But the Shagger protocols, they used a gradient gel 4-13%, thanks

ereyesaldrete
ereyesaldrete's picture
Hi Alberto,

Hi Alberto,

I am encountering the same problem myself. According to the Nature protocol you cite, on a typical gel apparatus setup, the chamber holding buffer closest to the negative electrode (I think this is the cathode, right? I always forget... :)) must also contain coomassie dye. Can I ask, how are you doing this in the phastgel? Sorry to answer your question with a question but I was hoping we can help each other out. Ihave a feeling that your drop in current is due to charge neutralization from the negative coomassie leaving your sample and accumulating in the anode. I will try running a native 4-15% phastgel, after soaking the native buffer strip on the cathode side in a 0.2% coomassie (10X from the recommended in the protocol as a starting point) solution. Wish me luck! 

Emilio