question about cAMP antibody

[lizhiwei]

As the molecular weight of cyclic adenosine monophosphate (cAMP) only around 200 Da, how come the band of cAMP of western blot is around 17KDa? and considering the small molercular weight , it is rather difficult to run the cAMP molercuular on the WB gel, how can I do that?

Thank you and headacher waiting your reply

[CharonY]

I am confused about what you describe. A western blot is made with proteins, not with small molecules like cAMP. Are you sure that you made a PAGE with cAMP?

[lizhiwei]

yes, this is what also confuse me. I am sure we can run PAGE for cAMP, based on the information from cAMP antibody company.... follow is the link:

http://www.abcam.com/cAMP-antibody-SPM486-ab24851.html

[CharonY]

Hmm, I see. However, I assume that the antibody is used to detect proteins that bind cAMP, rather than to detect isolated cAMPs in a gel.

While this is might in theory be possible (e.g. by running a sequencing gel, but only shortly) I cannot quite see an application for that.

 

Now to your initial first question in the OP.

A gel does not truly separate according to MW but rather according to electrophoretic mobility. The result is that in order to assess MW from a gel you need standards that are chemically similar (at least with respect to electrophoretic mobility) or you have to make them similar. An example are proteins that due to different amino acid compositions have very different charges and structures. In order to compare their sizes one has to eliminate (or reduce) these effects by adding SDS.

 

As such I am wondering, based on what as reference do you assume that cAMPwould run at an equivalent of 17kDa? Generally, such values are used for proteins gels (which cAMP is not, of course). Are you sure that you did not simply confuse cAMP with something else (e.g. CAMP, Cathelicidin antimicrobial protein)?