I know I'll probably sooner or later find out what's going on but I would appreciate if some of you (having more experience with this than me) could help me out.
I wanted to test the protein stability of proteins in 1 -10M urea. After making the dispersions (I used solid material to begin with) I centrifuged my samples and used the supernatants to prepare for protein measurement (EZQ). For this I added Leammli sample buffer containing 5% beta-mercaptoethanol (1:1) to the protein samples. Then I wanted to run an SDS PAGE to see protein distribution, but I read beforehand that samples containing urea should not be heated (or at least not over 37°C), so I thawed my already prepared ready-to-go samples at RT and loaded them onto the gel, 10µL per lane. In the picture below you can see lanes 1 through 6 are one protein set with decreasing urea conc (10 to 1M) . Lanes 7 through 12 are a different set of proteins and also in a decreasing order of urea used for these samples. I wonder why exactly it seems like my first set (lanes 1-6) isn't moving very far. Is it decreased protein mobility in gel? Even the marker on the far left seemed to be affected, however the marker does not contain urea! I know that I have more soluble protein in the first set, so I guess the urea (at a different concentration) is affecting the mobility of the proteins in different ways. Is that why I see a skewed rather than linear pattern? But I read that it's okay to use urea for SDS-PAGE. Or is it interfering at certain concentrations or messing with the SDS in the sample preparations?
I would really appreciate your thoughts on this, maybe someone has had similar experiences?
Thanks a million!
Nathalie, you are correct in that the different concentrations of urea within the same gel will affect the migration rate of your different samples through the gel - running gels in different salt concentrations will affect their mobility since you will be altering electrical resistance. As you run the gel you will probaly notice that the dye front also runs skewed. What is the question you want to address? Is recombinant protein less liable to cleavage in 10M vs 2M urea when lysates are made ?
The only way your gel will run without some distortion is if the salt concentrations and final ammts in all your wells is the same i.e the highest salt concn. or if the ammt you load is so small as not to perturb the salt concentration sufficiently to alter resistivity.The same will apply to your markers - they will not run true if your samples are in a high salt..
Ian
Lucrecia, have you used it? Can you buy them as precast and if so where? I basically just wanted to see if I can see on a SDS PAGE if some of my proteins in my sample were less affected by urea than others.
Nathalie.
We cast our own gels. I really dont know if they sell them precasted.
As In an sds-page proteins are unfolded...what are you expecting to see?
Regards
Lucrecia
Hi Lucretia
What percentage gel are you using (is it more than 12%)..higher percentages, though they can give you higher resolution, they can also affect mobility of your proteins. I have been successfully running samples with 8M Urea mixed with laemli buffer with relatively good mobility (see attachment) even after heating at 95 degrees for 5 minute. Normally after I have quantitated my sample, when I add lamli buffer instead of working from a 5x/4x stock solution, I prepare a 2X working solution. I would then add the buffer in a 1:1 ration i.e if I am loading 10uL of sample i would mix that with 10uL of lamli buffer. Am not sure why this works but my reasoning was that I am possibly diluting the urea and the beta-merk prevents further aggregation
P.S. These samples were resolved on a 5 %Stacking and 12% Resolving phase (I cast my own gels) at 80 V through the stacking and at 120 V until the dye runs out (these were not time...but it normally take more than 30 min) If you time your runs try not to time them for now...just let them run until the dye runs out as another reason for them not separating very well may be prematurely stopping the run
All the best
Nathalie, you are correct in that the different concentrations of urea within the same gel will affect the migration rate of your different samples through the gel - running gels in different salt concentrations will affect their mobility since you will be altering electrical resistance. As you run the gel you will probaly notice that the dye front also runs skewed. What is the question you want to address? Is recombinant protein less liable to cleavage in 10M vs 2M urea when lysates are made ?
The only way your gel will run without some distortion is if the salt concentrations and final ammts in all your wells is the same i.e the highest salt concn. or if the ammt you load is so small as not to perturb the salt concentration sufficiently to alter resistivity.The same will apply to your markers - they will not run true if your samples are in a high salt..
Ian
Sorry all, for just getting back to you now. I just saw that you all had commented on my question (besides Lucrecia, usually I get notified via email). Thank you all so much for your contributions! It all makes sense to me.
I have been used 1M urea as a finale concentration, boiling for 5 minute at 100oC, and the results was perfect.
Try using tricine gels. I also use around 8M Urea to solubilize my samples before loading it on the gel and they run fine. Tricine gels are known for better resolution.Good luck!
I know this is a case by case basis. My case is to dissolve spore coat protein in SDS-8 M urea at 100oC/70oC for 10 min and run on a precast 4-12% bis-tris gel (Invitrogen). I guess it works fine for me.
Tricine Gels resolve small proteins better, but not necessarily large proteins.
The electrophoretic mobility problem seen on the gel above is due to the imbalanced concentrations of Urea loaded into each lane. If you loaded different protein concentrations but kept the amount of Urea uniform, than the gel would have run better.
The other glaring issue is the empty lanes on the far right of the gel. If these had also been loaded with urea+loading buffer, at similar concentration, the charge irregularities across your gel would improve greatly.
the protein samples and the gels appear to be of very good integrity.
If you are boiling your samples with Urea, prior to loading, may result in carbamylation which can cause the fuzziness. consider not boiling after having solubilised in urea. Secondly you may want to precipitate your samples with Acetone or Methanol and resuspend the pellet in LB before loading you samples
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