Problems with SDS gel - Boiling samples, autoclaved runnung buffer, 4 layers (Feb/12/2009 )
I am having a real problem in running a protein gel.. today. I am wondering could this due to my mistakes: firstly, i had over boiled my samples, more than 10 mins at 90 degree Celsius. Secondly, I have mistakenly autoclaved the 10X running buffer and 10% SDS soln.
It took 1 hr for the loading dye to reach the separating gel at 80V, also the loading dye front did not linearized, it was like a big broad band. Anyway, then I turned it up to 180V and let it ran to the bottom. Surprisingly, the resulting gel had developed 4 layers: stacking gel, the separating gel including an Extra layer with the dye front, lastly, the bottom layer. It looked like as if the protein and the dye had migrated in this extra layer. So weird. How could this happened. Could this be the bufffer??
Also, Can you refreeze DTT denatured protein samples in -20??
Pls help
overboiling the sample could cause a problem, the protein could aggregate (then it would stay at or near the origin). you can incubate at 65C for 10-20 minutes to avoid this.
autoclaving sds is a no-no. if there is sds in your buffer then it will affect the run. your 10% sds solution is ruined.
so, your problem can be the buffer.
yes, you can store the denatured sample at -20C. you may have to heat treat it again when you use it or you may not (some do, some don't). it won't renature when it is frozen.
elysian_chow on Feb 12 2009, 04:21 PM said:
It took 1 hr for the loading dye to reach the separating gel at 80V, also the loading dye front did not linearized, it was like a big broad band. Anyway, then I turned it up to 180V and let it ran to the bottom. Surprisingly, the resulting gel had developed 4 layers: stacking gel, the separating gel including an Extra layer with the dye front, lastly, the bottom layer. It looked like as if the protein and the dye had migrated in this extra layer. So weird. How could this happened. Could this be the bufffer??
Also, Can you refreeze DTT denatured protein samples in -20??
Pls help
buffer could be the problem. Overboiling shouldn't make great difference. Usually 3~5min at 90C is good enough
how long and in what condition was your gel stored before sample loading?
Can we autoclave 10x of tris-Glycine buffer (without SDS)?
I have noticed that after autoclaving solution each time, some of the solution will lost via evaporation/boiling process. I just wonder if this will effect the concentration of the buffer...?
Also, I have noticed some white precipitation in my 1-month age 1x Tris-Glycine buffer (not autoclaved) stored in 4oC. I am not sure if it is fungus/mold or Glycine precipitation. Any idea?
dcch on Aug 28 2009, 09:56 AM said:
I have noticed that after autoclaving solution each time, some of the solution will lost via evaporation/boiling process. I just wonder if this will effect the concentration of the buffer...?
Also, I have noticed some white precipitation in my 1-month age 1x Tris-Glycine buffer (not autoclaved) stored in 4oC. I am not sure if it is fungus/mold or Glycine precipitation. Any idea?
i would filter sterilize the buffer.
loss of volume will affect concentration and autoclaving will probably also affect the pH.
was the white precipitate fluffy?
if not then it is probably due to some component precipitating (or crystallizing).
Thanks!
I will check back next time. I had discarded that buffer anyway.
The buffer only consisted of Tris and Glycine.