2 replies to this topic

[mik-i]

Ciao!

I am currently performing Western blotting on muscle samples. However, my results don't match for what I found earlier with immunofluorescence. For my protein of interest, in IF, I saw that it was upregulated in diseased tissue but that it was almost absent in normal control muscle tissue. However, in Western blotting on total extracts, both normal controls and diseased samples show the same amount of protein??? How can that be? The antibody is suitable for both IF and WB. I never had it before that something so different in IF (normal vs disease) is totally unchanged in WB?
Please could anyone help? I'm desperate  

Tnx!

[miBunny]

Do you know if you are working within the linear range on your western blots?  Have you tried diluting your input lysate by 3 or 4-fold dilutions to see if the signal fades similarly?  

Western blots have a very narrow linear range and it is very easy to saturate the signal.  You need to be sure that you are using an excess of your detection reagent and it helps if you have purifed protein to run a standard curve with.

[mik-i]

miBunny, on Oct 28 2009, 02:54 AM, said:

Do you know if you are working within the linear range on your western blots?  Have you tried diluting your input lysate by 3 or 4-fold dilutions to see if the signal fades similarly?  

Western blots have a very narrow linear range and it is very easy to saturate the signal.  You need to be sure that you are using an excess of your detection reagent and it helps if you have purifed protein to run a standard curve with.

Hi!

I'm afraid I didn't check that! I can't use the sample several times again and again as I don't have as much material (it's always a leftover of a patient's biopsy). So, I'm now doing a Western blot in which I'm at one hand using less sample and on the other hand diluting my antibody. So let's wait and see! Thanks already for the advice   !!! I also heared today that I could better do chemiluminescence, because then you can check the saturationgrade with certain software programs