I am facing difficulty to show separate bands for LC3A/B bands. I find only one band. Here are my procedures; I cultivated THP-1 (monocyte, macrophage both tried) cells with 2% FBS Media 24 hours,. I used M-PER and Cocktail Protease inhibitor to make lysate
1. Lysate (20 ug) Load on 4-12% NuPage Bis-Tris gel with MES buffer. Run 100 V, 90 min
(I also tried Bolt 12% gel at the same condition)
2. Transfer to PDVF membrane (0.2 um) at 35V, 2H
3. Blocked 5% Milk with TBST
4. Blotted LC3A/B, Cell Signaling (1:1000), overnight
5. Washed 3x5min
6. Blotted Secondary Ab (1:2000)
7. Washed 3x5min
8. Developed 2 min with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermofisher)
9. I found always one band.
The issue is that you are trying to separate low molecular weight protein bands that are close in size. Since 12% acrylamide is not giving you the resolution you need, I suggest you try higher % gels, which means you will have to pour your own, since such gels are unlikely to be commercially available. I've never tried to run protein gels above 12%, but I do know from running DNA sequencing gels, you can go as high as 20%. Since you are running Western blots - as opposed to staining and drying - this should not be a problem. Drying 20% gels is problematic, as they must be soaked in a glycerol solution prior to drying or they will fragment.
There is a lot of literature on analysis of high mobility group [HMG] proteins, which are of similar size to the proteins you are analyzing. I would search that literature to see their methodology for separating small proteins.
Hi Shahid try using a higher concentration of separator gel, 15% or more and run in a Tricine buffer system
As Oscar said, Tricine gels work very well in this case. I used it to separate proteins < 15 kDa and to discriminate by 2-3 kDa (in my case, truncated forms of my protein of interest). I used to make a layered gel: stacking at 4%, one upper running portion at 10%, and a lower running portion at 16%. If I remember well, 120 V for 2h should be enough - the running buffer should not get warm. The run is slower than in a 12% gel, but it's worth it.
Please, find the links below:
https://www.ptglab.com/support/western-blot-protocol/tricine-gel-recipe-for-low-molecular-weight-proteins/
http://www.hixonparvo.info/SDS4Peptide.pdf
The electrophoretic mobility of trycine anion is lower than glycine, so sepations of low molecular species are better performed in this buffers system
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