phospho-mapk & mapk as p42, p44 - double bands or just single band?

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Philipp
Philipp's picture
phospho-mapk & mapk as p42, p44 - double bands or just single band?

Hi, I'm western blotting on phospho-mapk and mapk.
Some antibodies show me the figure of one band or sometimes double while reading a paper.
what was that?

if double, the upper and the below are going together at the same expression level in western blotting?
I mean, I'm stripping the membrane after detecting the phospho-form first to re-detect the non-phospho form of mapk.

Otherwiese, is it possible to see if the upper only decreases while the below has no change in density? or vice versa?

Thanks

Omai
Omai's picture
Hi Phillipp,

Hi Phillipp,
Any time I've looked at Erk signaling (p42/44) I've seen it as a doublet in both phospho and total Erk. Any increase or reduction was seen in both bands (usually). I've never seen in the lab (or read in a paper) of a decrease in just p42 or just p44, and I always see Erk as two bands. Can you give the reference of the paper that shows it as one band?
Does this answer your question, or do you have a question more specific to your assay.
I used antibodies from Cell Signaling.
Omai

Arun Vaidyanath
Arun Vaidyanath's picture
 Hi Philipp,

 Hi Philipp,
I will go with the answer of Omai. I've also done the Erk activation both Phospho and non phospho(whole). Till date I also got two bands all the times. I am using the antibody for Erk from Cell signaling. There is slight variation in the band intensity with both bands, the p42 being strong when you compare in ImageJ, but I always use to get two bands clearly. Hope this helps you.

Britjade91
Britjade91's picture
Hi! I am carrying out western

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!