Not getting actin bands equal on Western blot - (Jun/09/2006 )

hi i am doing westerns and normalizing my proteins with actin but i have problem with that.i am not getting the same equal bands of actin.some are very dark some are very light and some totally disappears.i am upset becose my boss is not happy with that.pls help me and tell me some reasons and sloutions.thanks

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you have to tell a little more.
do you load different cell extracts? or is it the same cell type with different treatment?
do you determine the protein concentration in your sample before loading?

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thanks for reply
yes i am measuring protien concentration B4 starting experiment and i am using heart tissues.pls help me

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they are differnet treatments but actin should come the same in all the bands Bcos i am normalizing my proteins with actin

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i am using the same tissue type with different treatments

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two possibilities :
-either you are not able to load the same amount, the right volume. Check your pipet
-or your protein quantification is not correct.
do you dilute the standard in the lysis buffer? you should use the same buffer for the standard and the samples. do you have a bif difference in the protein concentration in your different samples?

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Have you tried probing for tubulin as a loading control instead?

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thanks for reply
-no i never tried tublin but i tried GAPDH and it is also little variation not a single band
-i am diluting with same lysis buffer which i am using for my samples
-i borow the pippet from some one to see the variations but same
-i am very carefull for my experiments not doing in hurry

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thanks
i never tried tublin but i tried GAPDH this time but not happy results mean not single band little variation.

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you may consider the fact of diluting in standard ddH2O instead of dilutiong in lysis buffer that may interfers with your OD measurement and/or bradford assay.

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