Protection Lanes? - (Dec/16/2011 )
Hi all. I was hoping to get an opinion on a problem I am routinely encountering with the boss.
He insists that the last lanes of a loaded gel should contain some throwaway cell lysate as a "protection" lane. Simply adding buffer to equal volume is not good enough in his mind, as buffer does not contain protein, and without protein he insists that you will get screwy results. The problem is that those two lanes that get spent are oftentimes precious and could be put to better use as real sample or duplication, and I cannot see any rational basis for having protein in an adjoining lane.
To be clear, this is not a control. We don't care about the staining of this lane, only the presence of protein to assure a good run. If something goes awry with a protein band and no protection is there, the boss insists that it is due to the lack of this protecting lane and nothing else, which seems to be ignoring other potential sources of error.
Has anybody heard of this practice? Do you think it is worthwhile?
deadally on Fri Dec 16 16:30:50 2011 said:
Hi all. I was hoping to get an opinion on a problem I am routinely encountering with the boss.
He insists that the last lanes of a loaded gel should contain some throwaway cell lysate as a "protection" lane. Simply adding buffer to equal volume is not good enough in his mind, as buffer does not contain protein, and without protein he insists that you will get screwy results. The problem is that those two lanes that get spent are oftentimes precious and could be put to better use as real sample or duplication, and I cannot see any rational basis for having protein in an adjoining lane.
To be clear, this is not a control. We don't care about the staining of this lane, only the presence of protein to assure a good run. If something goes awry with a protein band and no protection is there, the boss insists that it is due to the lack of this protecting lane and nothing else, which seems to be ignoring other potential sources of error.
Has anybody heard of this practice? Do you think it is worthwhile?
I've heard about loading a 'protective' loading buffer in the side lanes to avoid non-straight lanes. The rationale is that if there is a gap, protein would have the opportunity to run sideways and not straight down. If you have loading buffer in both sides, you 'force' the middle lane to run straight down. Is this true?
It is false, I routinely run gels without any protein or loading dye in the outside lanes, or even run crucial samples in the outside lanes, and very rarely have a problem with smiling or lanes spreading. If you have your running conditions correct, there should be no problems!
bob1 on Sat Dec 17 21:03:38 2011 said:
It is false, I routinely run gels without any protein or loading dye in the outside lanes, or even run crucial samples in the outside lanes, and very rarely have a problem with smiling or lanes spreading. If you have your running conditions correct, there should be no problems!
There's alot of myth going around that proteins transfer differently depending on their position on the gel.
I also noticed that my protein ladder on the far left is always distorted compared to a similar ladder run on the other end.
But I do know of people who run alternate lanes, e.g. empty-sample1-empty-sample2-empty-etc... just to give a nice separation and avoid merging bands.
science noob on Sun Dec 18 03:10:43 2011 said:
There's alot of myth going around that proteins transfer differently depending on their position on the gel.
I also noticed that my protein ladder on the far left is always distorted compared to a similar ladder run on the other end.
But I do know of people who run alternate lanes, e.g. empty-sample1-empty-sample2-empty-etc... just to give a nice separation and avoid merging bands.
Transfer rate can be affected by the presence of other gels in the same transfer tank (in my experience), such that the membrane that is furtherest away from the black electrode seems to transfer slower than the one closest. In terms of physics, I can't see why this would happen, but it seems to be a reality.
I very very rarely have problems with merging/distorted bands working with proteins from 16 - 130 kDa, there must be something about the set-up or running/transfer conditions that you are using that causes this to be a problem. The only time I get a distorted lane is when I can't lay out a 6% gel evenly without it tearing, even then, the bands are still discrete and sharp, just that there is a bend in the shape of the lane so that the bands are not directly over one another.