Hi,
i had a problem recently in SDS gel which i ran. The width of the lane loaded with marker protein got shrinked, while the other lanes are ok. I guessed it might be because of the low amount of marker I loaded (5 microliter) compared to 60 microliters of the sample in nearby wells. Is it the real problem? Can i add some extra sample loading buffer to the marker which might make the volume of sample in all wells equal. Comments please.
Thank you.
Ya i think you rite ...... For SDS gel minimum amount of sample to be loaded in a well is 25 microliters ... try this out...
protein bands are not always depends on sample loading, if you have sufficient concentrated sample. in this case i feel you must load your sample first than marker because some times what happen sample may get diffuse if you keep for long time before running PAGE. cause less vol of marker you can load immediately. you just try, and one more thing give sufficient time for sample to be settled down at low % gel n then run for higher % gel with higher voltage. all d best.
Thx Rajdeep, i have loaded the marker only after loading sample.
acetone precipitates the proteins leaving many of the other substances in solution so not only do you concentrate (by redissolving in a smaller volume) but you also get rid of many ionic impurities which can interfere. Going back to the original post, I believe that high salt in the samples which is not present in the standards is what caused the standards lane becoming narrower.
Yeah, we precipitated it with Ammonium sulphate...but not with acetone....And Nick, you might be correct the salt in the sample might be a problem....next time i will be more cautious in dialysing the sample to remove the salt....
I agree with Alberto and Nick, but -I'm sorry- I disagree with Jamuna. For gel-electrophoresys there is only a maximum (not minimum) loading volume, given from the gel dimensions and wells. For a good run, follow the Alberto-Nick indications, and let resuspend the precipitate samples in the same volume of loading buffer (I tried 5-10ul successfully).
- Badges
- Science method