i am using the protocol written by Britta Blumenthal (2011) on a bacterial transcription factor. My lanes are smearing and getting stuck at the stacking/separating interface. How do I improve my resolution? I run for 30min at 150V (15mA) then 250V for 2-4 hrs (2-4mA). Gel is 4-15% gradient with a 3.2% stacking.
Probably best thing I can suggest now is to check the pH of your buffers used to prepare the gel and stacking gel. If you're using old reagents, then I suggest to prepare fresh. Sometimes preparing fresh buffers helps a lot in troubleshooting
From my experience, there are some factors that may affect the separation: The concentrations of gel, buffer and the substance of your interest, and current used for stacking vs separating gels. The electricity may also affect, 150V is too high may increase the temperature; try to use less than that at the first stage for several minutes. Make sure the gel is fresh and not dry. What I can see from the photo is the gel concentration or preparation time and method which make the gel solid more than it should be.
If something is getting stuck between the stacking and separating gel interface, means you need lower concentration on the upper gel limit. Usually happens if you have large protein complexes (> 1 MDa). I would recommend a 3-12% gradient gel.
Smearing can come from overloading the gel with protein, cell contaminats (DNA, lipids) or excess detergent. Since you are working with a transcription factor, just in case, for BN page avoid high Mg or Ca concentrations (>1 mM) as these can cause dye precipitation.
Are you working with a purified protein or cell lysate?
Christian Magaña Vergara i am working with a purified protein. My protein is only 30kDA but can also dimerize or tetramerize. the protein is also purified with anion exchange before affinity purification to remove DNA.
Hey, I was also facing the smear problem initially when I started running the BN page for membrane protein samples.
I solved it by adding 500mM 6 aminocaproic acid while casting the 4-15% gels. Also, I added 500mM 6 aminocaproic acid to the cathode buffer(both with and without dye).
This solved the smear problem as the protein got solubilized better in the gel.
Moreover, run the gel at 100V initially until the samples leave the wells and enter into resolving and then increase the volts to 200V and run for 2-4hrs.
Hope it helps
You can refer to BN-PAGE nature protocol for further insights; doi:10.1038/nprot.2006.62
Hope it helps.
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