Tricine SDS-PAGE
Claire Jennings
Monday, 01 June 2009 22:02 UTC
I’ve recently tried running tricine SDS-PAGE gels using the protocol described by Schagger (2006). It’s a great paper but after setting up and running the gels on several times, I keep finding that my protein samples can migrate through the 4% stacking gel but not into the 16% acrylamide resolving gel. I have re-checked my acrylamide concentrations and powerpack conditions. Any suggestions on how I might solve this problem would be much appreciated.
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What size protein are you trying to separate?
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It is a 28 kDa protein. I’ve heard about the increased resolution of tricine gels and would like to see if i can resolve mass differences due to PTMs… I think this technique might not resolve such small differences but would like it to work so i can see if this is the case or not!
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In general Tricine gels are great for resolving peptides. What sort of PTM are you looking for? Are looking for a general addition of mass or are looking for a site specific addition. Just want to have as much information as possible.
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I’m not really sure how the protein has been modified yet as waiting on MS results. Its possible that the protein maybe be affected by non-enzymatic glycosylation (glycation) but other modifications could quite possibly be present. The purified protein preparation shows only one band on a laemmli PAGE gels, but is shown to be heterogeneous following separation on a cation exchange column. The samples I applied to the tricine gel were all taken from fractions eluted from a cation exchange column so there are charge differences between each of the protein isoforms according to my chromatographic results.
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Hi Claire,
I use Tris-tricine SDS-PAGE gels on a regular basis to get the best separation and resolution in the 5 to 15kDa range. In my hands, molecular weights higher than 20kDa do not seem better off in tricine versus glycine, etc.
The setup of my gels is as follows: I either pour (or purchase) a standard 15% Tris-HCL gel, include 1% SDS in my sample loading buffer, and then use a cathode buffer that contains Tris, Tricine and SDS and an anode buffer made of just Tris. -
Hi Elizabeth – That’s good to know about your results with proteins over 20 kDa. My current setup is pretty similar to yours with respect to buffers etc. Do you add glycerol or urea to your Tris-HCl gels? Thanks very much for the information.
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Generally my PAGE gels do not contain glycerol, urea or SDS since I already have denaturing agents (i.e. SDS) in my sample buffer and cathode buffer and I heat my samples to 95C for 5 minutes before running them.
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Do you use the same sample loading buffer when you run your Laemmli gels with your purified protein and your Tricine-SDS gels after the chromatography of the purified protein? Is the one more denaturing than the other?
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My sample buffer is at 2x and contains: 0.1M Tris, pH 6.8, 24% glycerol, 2% BME, and 1% SDS
There is no dye because I stain the gel with Sypro Ruby and dyes like bromophenol blue interfere with the visualization of my proteins -
I was having the same problem as before. After I pour my stacking gel, I pour a layer of 75% EtOH on top to make the resolving gel smooth and bubble free. I was taught to just remove the EtOH before pouring the stacking gel. However, this caused problems, and nothing would get into the resolving gel. Now, I remove the EtOH, then rinse thoroughly, and dry the resolving gel thoroughly before applying the stacking gel. Hope that this helps.
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