Hi all
I am having trouble with detecting phospho-IGF-1R by Western blot.
I am trying to detect both the total IGF-1R beta and phosphorylate IGF-1R in MCF-7 and HEK293 by IP and Western blot.
I usually first do IP for the total IGF-1R beta (500ug of total protein from cell lysate) followed by Western blot for both total IGF-1R beta and phosphorylated IGF-1R beta.
I use NP-40 lysis buffer to lyse the cells. The pimary antibodies I use are
Rabbit polyclonal anti-IGF-1R beta (sc-713)
Mouse monoclonal anti-phosphotyrosine (4G10)
I block for 1 hour at RT with Odyssey blocking buffer (OBB-TBS) and incubate with primary antibodies dissolved OBB-TBS in in a cold-room overnight.
I personally thought if you want to detect phosphorylated proteins, TBS is always an choice. However, the protocol has never worked for me to detect p-IGF-1R, let alone the total IGF-1R beta.
Any advice is appreciated.
Hello Yinan,
My first thought is: If you can not stain the total IGF-1R, then most likely your pull-down has not worked. Or do I misunderstand? Could be your AB is not suitable for IP. Also you can try the other way arounf by pulling down p-IGFR. Is there a special reason you are doing IP instead of WB on whole cell lysate?
Hi Thomas.
I once did WB on whole cell lysate without IP, but I could not see specific protein expression for IGF-1R on a membrane at the location where it is supposed to be. So I thought I should pull out the total IGF-1R first by IP and then blot for p-IGF-R. Am I wrong to think this way?
Hi Antimo
You mean IGF-1R?
Molecular Weight of IGF-IRα is130 kDa and molecular weight of IGF-1Rβ is97 kDa
However, molecular weight of pro-IGF-IR is 200 kDa.
Hi Antimo
I always add 2-mercaptoethanol so the sample should be in a reducing condition, meaning that the disulfide-bond in IGF-1R is broken down, so there should not be 200kDa band
Hi Yinan
Sometimes the binding of the dimers is so strong that not even the mercaptoethanol is able to break it, I believe me I do western blot are ten years and I've learned not to believe the only protocols
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