Troubleshooting for BN PAGE - (Jan/27/2015 )

Hi,

 

I am trying to run Blue Native PAGE for cell lysate using following protocol-

 

Separating Gel gradient- 5 to 13.5% acrylamide

Stacking gel- 4% acrylamide

 

Sample buffer-Bis Tris-100mM, 6 amino caproic acid-500mM, 30% glycerol and CBB G250-5%

 

Cathode Buffer(1X)-Bis Tris-15mM, Tricine-50mM and CBB G250-0.02%

 

Anode Buffer (1X)-Bis Tris-50mM

 

I am doing everything at cold conditions but my gel does not run after the dye front reaches 3/4th. According to the protocol( published in 2006), I run the gel at 100V until the protein enters separating gel, then I increase the voltage to 180V . But it runs only for 30 min then even for 5mA the voltage exceeds to 300V (in BioRad, thats limiting) and I get Error 1 and it stops. I have tried changing everything. But Don't know where I am going wrong. Please help me!

are you sure that you can't increase the limit to 500 volts?

 

in looking over the protocol for bn-page, i see that bis-tris has been replaced by imidazole (bis-tris interferes with protein determination assays). maybe it will work better for you if you use imidazole.

 

also, make sure you follow directions for adjusting pH (or not, as recommended).

Thanks mdfenko! Imidazole needs to be replaced in both sample buffer and cathode buffer? But do you think bis tris could be the reason for stopping my gel in between? pH, I have adjusted 7 for all the buffers as mentioned in the protocol. Can you share the recent protocol?

 

I am going mad, not able to figure out whats happening  

 

BioRad power pack that we have, has limit of volts max 300 and current 400mA.

i found the protocol using imidazole here: nature protocols

 

(i'll attach the article in case you can't access the full text).

 

we always use power supplies that output at least 500 volts (most will output 1000 volts). as the gel runs and buffers deplete, resistance increases. you can either allow higher voltage or change buffers during the run or use a different buffer system.

 

sometimes the buffer(s) may be ruined by adjusting the pH (overshooting and recovery, bad electrode, bad technique, etc) and will not operate as advertized. you have to be very careful to follow the protocol exactly (at least until you have enough knowledge and experience to safely modify the protocol).

 

 

Thanks again Mdfenko!! Its a great help. I read the protocol, they have also mentioned that in cathode buffer when we add coomassiae G250, it should not be stored at 4 degrees , as the dye has the tendency to aggregate and that can interfere with protein to enter in the gel . May be here also I am doing wrong, because every time I would keep the buffer in cold. I need to re do everything with some modifications, Hopefully it will work now. Will definitely update you if it works

Hey mdfenko, My BN PAGE is now running properly. I used the nature protocol, replaced bis tris with imidazole and prepared samples in buffer which contained Glycerol, CBB, Triton-X-100 and imidazole. For single proteins, I get very nice resolution but again for protein complex, I see smear or streak. There may be few bands also but you have to see carefully then only you'll see them. Can you advice how do I imrove the resolution for my protein complex? I am loading whole cell lysate from plant. Attached the gel picture. 

when you run a lysate or total extract there are a lot of proteins, complexes and aggregates.

 

a couple of things which may improve results, either individually or in combination, are:

 

allow the sample to "age" in the sample buffer (since this is a native page you shouldn't heat the sample to speed the process)

 

add enough reducing agent to break up intermolecular disulfide bonds (this may break up complexes that you want to preserve)

 

remove aggregates and particulates by centrifugation and/or filtration (this is the first thing i would try if i didn't try either of the previous and would always add it to them)

 

use a staining method specific for your complex (eg western blot)

Hey mdfenko,

 

I tried the things that you suggested. But Recently I got to know, that, my protein complex has lot of RNA in it . Would that be a reason to get all the streaking ? Is there any way, by which I could remove this RNA? Since the complex could be transient, I don't want to give any harsh treatment. 

rna of varying length could cause streaking. a specific rna molecule would cause a shift in the band (or two bands).

 

this paper gives a method for dissociating rna from protein:  Dissociation of nucleoprotein complexes by chaotropic salts.

 

if the rna is not bound to the protein then you may want to try a rna purification method to separate them.

Thanks mdfenko! paper is useful. But I was wondering, if we dissociate RNA from the protein complex, won't the complex change then? . And here there are adding salt to dissociate RNA,that might change everything?