Western blot analysis

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Britjade91
Britjade91's picture
Western blot analysis

Hi! I am carrying out western blot analysis for the first time, on phospho-ERK amongst others. I wondered, what is your method of analysis for these?

I am currently using ImageJ but am experiencing problems;
- I can only select an area of minimum dimensions around an area of interes, which is larger than I would like
- one of my blots is "dirty", so I find there is a lot of background interference which I cannot resolve
- I am unsure what area to select in the lane selection - are there any rules around whether the background cut off selection must be straight?
- can I analyse both of the doublet bands in the same plot? (as shown in attachment?)

Thanks for any help!

Chin Fen Teo
Chin Fen Teo's picture
Hi Britjade91,

Hi Britjade91,

What is the blot corresponding to the imageJ histogram? phospho-ERK? Does the blot give you one or two bands? Depends on the source of your sample, some time you see one band, some time you see two...

If you are getting two bands and both of them are your target, then I would grab the area for both peak. If you are sure that one of the minor band is some non-specific band, then I would just draw a line that only include the major peak and omit the minor peak.

Hope this helps.

nktcells
nktcells's picture
Hi there! 

Hi there! 
I would actually quanitfy each band separately. I've seen a decrease in p44 and not p42 and vice versa, so to circumvent that, try to quantify them separately and then add it up. 
It's hard to tell why your blot would be dirty, but try to put your secondary in milk. 
Best of luck!