Dear Jérémie Courraud,

One possibility is that your sample proteolized. do you use protease inhibitors?

Other possibility is the amount of protein? probably you need more sample to detect higher molecular proteins.

Didi you stanined both gels concentrator and separator gel? to detect proteins in concentrator gel?

Stain also with silver is much more sensitive than coomassie blue or Ponceau

Best regards,

Jérémie Courraud This is one guess. If you are using semi-dry blotting, your device might be out of order. The current has stopped flowing in some area of ​​the electrodes, and so on. How about this?

Probably not, because the proteins upper 79 kDa are not visualized in gel. It is important confirming running in gel, after immuno-electron-transference to membrane, corroborate the correct bands and immunoblotting.

One guess is that the large proteins were not transferred to the membrane. You may confirm the presence of the large proteins on the SDS-PAGE gel by staining before protein transfer. If they appear on the SDS-PAGE, you may need to increase protein transfer time or use different type of membrane.

Dear all,

First thank you all for your tips and proposal, I like to see that people help each other in science :D

I'll go one by one :

- Nap : we checked for the samples that work well with a technician but not with us and we also took gels from another team (so other reagents)

- Gladys : Yes proteases and phosphatase inhibitors and I'm actually over-expression from a plasmid (CMV promoter) so I usually get enormous signals. As I said for the coloration we did ponceau and blue and we can see the "protein ladder" bellow 70kDa and a nice white membrane/gel on the top

- Michitoshi no semi-dry saddly, This would save me so many time but thank you for the idea :)

- Jainmei : the problem is 100% during migration, we even see ladder disapearing after a certain among of time.

To give you all some news, I just did my Western in another lab that loaned me the material for the whole process (doing the gel, migrate, transfert, etc ...) and it worked perfectly well so I'm actually running here and try to find the problem of our lab.

Once again thank you all for your help, I'll up-load this post as soon as I find the origin.

Best to all of you

Jérémie

Because you've apparently been doing these blotting experiments for four years, I'm inclined to believe there's a problem with the gels. Where do you get your gels? Have you confirmed they are running as expected? When you figure this out, please let us know what the problem was.