hi

You put tanks on ice during gel running, If that does not work, you can work on lysis buffers.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020342

http://cshprotocols.cshlp.org/content/2015/3/pdb.rec081539.full?text_only=true

https://sites.google.com/site/kittisakyokthongwattana/lab-protocols-recipes/isolation-of-chloroplast-envelopes-from-dunaliella

good luck

As Reza suggests use ice.. Which voltage/Milliampere you run it ? This could be too high..

Thanks for the suggestions. I'm running the gel on ice already. I start at 60V (around 4-5mA) and increase later to 75-100-150-200V (but the mA always stay around 4-5).

Voltage seems okay.. I attach you the BN-PAGE protocol from nature they have a troubleshooting section. One problem might be the solubilization step where the ratio of protein and detergent is not okay...

By the way just to get it clear..  Do you expect only sharp bands in your gel ? I never experienced that. Some proteins especially those which are complexes of more than one protein will always give you a broad band as some parts might dissociate and so on.. I suppose it lies within the method of working with non-uniform proteins in BN-PAGE oppposed to uniformely denatured proteins in SDS-PAGE. Just look at this gel picture published. Its just a theory without proof.

http://www.nature.com/pr/journal/v50/n5/fig_tab/pr2001236f5.html

Of course I'm not exspecting to get sharp bands only. But I don't understand why this upper band is "breaking". By the way, maybe it's not especially about this band, because in the meantime I repeated the BN and this time the gradient was much better, so that the band migrated deeper into the gel. But this "breaking" occured again at the same position in the gel. So maybe it's a problem with the gel itself?

The upper green band ? Do you cast the gradient throughout the whole gel or do you have a "collecting gel" above the gradient gel ? Maybe just some stuff is precipitated and won't enter at the intersection between the two gels. I experience that as well. But just a guess

hi

you must add Protease Inhibitor Cocktail to cell lysis buffer , because almost protein lysis and breaking occurred by cell protease enzymes action during cell laysate preparation.

http://www.sigmaaldrich.com/catalog/product/sigma/p9599?lang=en®ion=IR

good luck

Sorry, but yes, I'm talking about the first thick green band (PSImonomer/PSIIdimer). I'm casting the gradient gel first and add a stacking gel after polymerization. I add water or isopropanol on top of the gradient gel for polymerization so that unpolymerized stuff should be washed away, and also I'm washing the wells with cathode buffer before loading.

Okay.. What is your gradient Acrylamide %. And what is the stacking gel percentage ? If the "change" in percentage is too high some proteins will not enter the gel.. Or some of your stuff precipitates (is precipitated) ?

My gradient is 4.5-12.5%, the stacking gel is 4%. So this should not be the problem. But I think I solved it now: When casting the gel I used less APS and TEMED to let the gel polymerize more slowly. The "breaking" almost completely disappeared. So if anyone else has this problem I suggest following this hint. Thanks for all the helpful suggestions!