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Lane spillover (?) on Western Blot


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#1 Maroulis

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Posted 07 January 2016 - 11:03 PM

Hi everybody,

 

I run recently two 12-well precast gels (Bio-rad) at the same time. I loaded 20 ul of sample in each lane (max. allowed) and I did it relatively slowly in order to avoid getting sample out of the wells while loading.

 

When I blotted for Actin (45 kDa) the next day, I noticed that the bands from individuals lanes were sort of connected with each other by small, curved lines like ears. I did not get the same pattern for histones (low MW) though.

 

Could the observed pattern on the actin blot mean a spillover between the lanes? I have attached an image showing the blot.

 

Thank you very much in advance,

 

Aris

Attached Thumbnails

  • Actin blot.jpg


#2 mdfenko

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Posted 08 January 2016 - 04:44 AM

not so much "spill over" as diffusion of sample into the "teeth" (walls?) of the wells. although, you may have had a little spill over that sat on top of the teeth (if the teeth were recessed from the top of the plate.

 

to avoid diffusion you should load more rapidly (you may have to load a smaller than maximum volume) and start the run immediately after loading.


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#3 Michael Starr

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Posted 18 March 2016 - 12:29 PM

I've had this issue before. A higher rank scientist saw it and said it was spillover. But you say it could be diffusion into the teeth, as well, which I haven't heard before. Doesn't diffusion take a very long time though? And if you don't notice any of the sample (which has bromophenol blue in it and is thus able to be seen) spilling above the teeth when loading the wells, how could spillover happen?



#4 mdfenko

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Posted 19 March 2016 - 03:17 PM

you can have spillover through voids (or bubbles) between the teeth and the plate. it doesn't have to go over.


talent does what it can
genius does what it must
i do what i get paid to do




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