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Unequal loading even after corrections!

Unequal loading even after corrections! - Western Blot Forum

Unequal loading even after corrections! - 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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Old 10-28-2010, 08:23 AM
Pipette Filler
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Default Unequal loading even after corrections!



Hello all,

I have very difficult problem to which I need your help! I have whole cell lysates from different human lymphocyte cell suspension which are diluted in 2,5 X Laemmli buffer (Laemmli includes NaF & Na3VO4). After collecting my samples I have stored them in -20. Before loading the samples into the SDS-PAGE gel I have sonicated them to break the membrane (3 x 10 seconds with 7,5 amplitude) and then measured the protein concentration by RC DC kit (BioRad). Then I have loaded equal amount of protein in the well, so that in every well the loaded volume is a bit different depending on the concentration. Result is unequal loading, I am using b-tubulin as loading control. I have tried to correct the loading by calculating the b-tubulin signals and then calculated how much more or less I need to load my samples. But even after these corrections I end up with very unequal b-tubulin response. I have tried this many times but every time the result is the same. I have tried to load with different kinds of pipet tips and pipets but nothing helps.

Does anyone have similar problems and how have you solved them?
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Old 11-05-2010, 06:24 PM
Pipette Filler
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Default Re: Unequal loading even after corrections!

I don't have your problem with uneven loading....but, I was taught to load equal volumes into each well....adding my protien concentration to a volume of H2O that makes up to the volume I need....example: Want 10uG total protein, Sample one is 5ug/uL take 2uL of sample and 8 uL of H20, Sample 2 is 4ug/uL take 2.5 uL of sample and 7.5uL of H20....My sample buffer is 2X so add 10uL of sample buffer to each sample tube and load 20uL per well..may be worth a try
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