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Help!! Uneven actin expression .


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#1 Lexitus

Lexitus

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Posted 02 July 2010 - 06:57 AM

Hi good day,

I am a student who has just started to do western blot during my Internship. I am facing a major problems which is my actin expression came out unevenly and some thin some thick. I have been redo the whole things many time with using the same protien sample but the expression turn out almost the same. I have done protein assay each time prior loading the samples.
Please help me to find out what I did wrong? Pipetting technique wrong?

Thanks extremely much :D ;)

#2 krisztina

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Posted 08 July 2010 - 02:54 PM

Hi

You should try another loading control especially if the uneven bands are consistent with your samples (for instance the same treatment always causes thicker bands).
Actin is a common, but not at all useful control for every situation. For instance when I do a treatment with soluble reagents, I get even actin on samples, but the same reagents coupled to beads cause uneven bands (due to actin change upon treatment).

You could try tubulin, GAPDH or even another protein that you know does not change under your conditions.

Try and stain your gel either with Ponceau before immunodetection or Commassie after you no longer need your blot. If the bands are fairly even you certainly should go for a new loading control.

Good luck,




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