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Ladder problem

Ladder problem - Western Blot Forum

Ladder problem - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 07-03-2012, 09:13 AM
Pipette Filler
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Default Ladder problem



Hi guys,

I encounter a problem almost everytime I do a WB; my ladder seems to 'dilute' adjacent slots. I don't know why it is doing this. I always cast my gells following protocol, and 'empty' slots I fill with an ammount of 4x sample buffer comparable to the ammount of 4x SB in other slots.
Could the problem be that the ammount of protein that I load is too high? I always load ~40ng (20uL) of protein. The gells that I cast are 15% acrylamide, and I use a 10 slots comb.
The ladder that I use is bought from Fermentas (Thermoscientific), it's productnumber is 26616. I load 5uL of ladder each time I do a WB.

Has anybody else encountered this problem? Or does anybody know why the ladder is spoiling my results?

With best regards,

Dennis
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Old 07-05-2012, 09:00 AM
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Default Re: Ladder problem

By "dilute" into adjacent slots you mean seeing faint bands of the ladder into the adjacent slots?
In that case, maybe you pour off accidentally some of your ladder to the adjacent slots. Sometimes bubbles inside the wells harass the loading of the samples and drag some of the loaded quantity out of the well, and most prolly into an adjacent one.
Two quick ways to get rid of the trapped air bubbles is either to go with the needle of the syringe and remove them or pouring some ethanol straight into the well.
Some other scientists also increase the percentage of glycerol in the sample buffer in order to ensure perfect loading in each well.

Now if you dont have any overflow and subsequent loading of the ladder into the adjacent wells, then you might have not a good current flow across your gel, or across the wells that you load your ladder (voltage inconsistency across the gel). A quick solution for that is simply load the ladder in the opposite side of the gel. If the problem persists, please upload a photo of that gel.
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  #3  
Old 07-05-2012, 09:42 AM
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Default Re: Ladder problem

try 2.5uL of the said ladder would be best if you show us the gel
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