hi Dominic,

does it always cut out after about the same time in which case there may be a pre set limit on either time or voltage x current. Check the power supply is set to run indefinitely

good luck

paul

Thanks Paul, but yeah the power can run indefinitely.

Hi Dominic,

Are you using 2 running buffers for BN-PAGE. One colored blue with CBB and the other colorless. Do you mix your samples with the right CBB sample buffer. Check out the CBB. One is G 250 and the other is R 250. The CBB gives charge to these proteins without dissociating them. Once the blue color of Buffer 1 reaches 1/3 rd of the gel replace it by the colorless buffer. Run these gels at 6-7 degrees at low voltages. It takes almost 4-6 hours to run these gels. run them on fixed voltages. You can see the current slowly going down as the gel runs.

You will have to stain and then destain it for long time to get clear bands.

Good luck

Mritunjay

Mritunjay Bhaiya,

Yes, i am using 500mM tricine+150mM bis-tris+0.02% CBB(G-250) for the blue cathode buffer and the same without CBB for the colourless cathode buffer and 500mM bis-tris for the anode buffer both pH 7.0.

Yes, i use CBB(R-250) in my sample buffer.

I run my gels in the cold room at low voltages i have tried starting with 30V,50V and like you say i have seen the current dropping with time, for e.g Yesterday i started out with fixed voltage at 50V this displayed a current fluctuating at around 2-3 mA at about 30 min into the run i noticed the current was fluctuating at 1-2mA and at 1hr 17min the run just stopped.I changed the voltage to 70V and again 2hr 20 into the run it stopped again!!

My staining and de-staining is not too bad actually just the shape of the bands is not pretty, like really curved, you know in a frown!! hence i think the run was too fast but how can i slow it down further when the run keeps stopping?

i am thinking of changing things up and see what happens i am planning to set up a run starting at 100V and then halfway through the run raise it to 500V like in Schaggers original protocol... i dont know lets see...

Hi Dominic,

You can also try running these gels at the bottom of 4 degrees fridges with the power pack kept at the top (outside) of the fridge. Sometimes the power packs are not compatible with cold room.

Hi

Maybe you should avoid the Tricine in the Anode buffer in the original Nature protocol (attached) , Tricine is only in the Cathode buffer (also in Tricine SDS) which "carries" the charge as far as i can remember.. so maybe double check your protocol. I attach the nature protocol...

And why you use R-250 in sample buffer ? I only mix glycerol with cathode buffer and load this after i poured in the cathode buffer...

Good luck

Thanks Norbert.

I already do not use tricine in my anode buffer, Only in the cathode buffer as you have rightly pointed out.

I use coomassie in my sample buffer, it binds the protein, causes a charge shift of the sample protein to the negative side which enhances migration through the gel. personally i have used both G-250 and R-250 and there is not much difference. My cathode buffer has 0.02% coomassie i think it might be too little with further dilution on adding to sample... plus, i also have aminocaproic acid in the sample buffer to help keep the protein in solution which i dont have in the cathode buffer...

Thanks for the nature protocol by Wittig et al from Schagger's group in Frankfurt.I had it already, along with Schaggers 1991 Anal Biochem article with Von Jagow. attached.

Might be nice to know the higher voltages aren't too bad actually, i got some okay bands with that, so yeah, and my run was continuous, no premature termination.

Just need to get some more protein samples and i think i just might get pretty bands!

Thanks again everybody!

cheers!

Hi,

I forgot to add. sometimes too much salt concentration could give you wavy bands too.

Mritunjay

I am actually working with mitochondrial membrane proteins and to solubilise them without loosing their activity i got to use a mild detergeant, nonionic ones like maltoside/triton x-100 (i use maltoside) but the thing is these detergeants don't work too good in low ionic strength solutions (atleast for mitochondrial membrane proteins). Some guys add NaCl others both NaCl and aminocaproic acid others still not only NaCl and aminocaproic acid but also imidazole which is amphoteric and highly polar to raise the ionic strength and or enhance solubilisation of the protein.

Again, you see, Chloride ions have higher effective mobility than proteins on an electric field so the salt separates from the protein raising chances of protein concentration/aggregation, reducing mobility in the gel and a probably not so pretty run...

So yeah, anyway i use only aminocaproic acid in my sample buffer but quite alot of it (1M) so as to enhance and maintain protein solubilisation...

Greetings,

If you already try to use the fresh cathodes (even fresh anode) and it does not help, it could be just simply changing your top chamber or power pack to see... Also make sure that there is no leakage of the cathode from the top chamber into the bottom part. Sometimes, a very slow unobvious leakage can cause that problem too... I do not think it's the buffer thing but rather the way things are set up.