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How can I separate LC3A/B I and II bands on western blot?
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+ Thp-1
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+ Fetal bovine serum
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Neil Farbstein
How can I separate LC3A/B I and II bands on western blot?
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.
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.
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.
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.
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
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
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The electrophoretic mobility of trycine anion is lower than glycine, so sepations of low molecular species are better performed in this buffers system
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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Hi Shahid try using a higher concentration of separator gel, 15% or more and run in a Tricine buffer system
Hi Shahid try using a higher concentration of separator gel, 15% or more and run in a Tricine buffer system
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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.
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.
More
VOTE