Wierd blot behavior

[eventhorizon]

Hi,
All of a sudden my WB has stopped working only for anti-rabbit antibodies:
I checked the following:
1. THe transfer is fine
2. All mouse antibodies showing good signals
3. However rabbit antibodies do not show any signal even for abundant target proteins
4. Used tested primary and secondary rabbit ab, tried even brand new arrival of both primary and secondary rabbit antibodies.
5. The target protens are all expressed in the cell-lines I tested.
6. The ECL substrate is brand new and worked great for mouse
7. All conditions same but rabbit antibodies just WONT work.


Can anyone please explain this sad happening in my life?

[Anastasia_S]

Strange...
Have you tried contacting your supplier with this question, maybe they have come across anything like that?

[Anastasia_S]

When you say rabbit Ab, do you mean anti-rabbit or produced in rabbit?
Just a guess - maybe something changed in your incubation soln (like new water filter, new batch of something) and you are getting an antigen or something with similar conformation that takes out your rabbit Ab before it binds to your protein on the membrane.

[eventhorizon]

Hi Anastasia
Yes, I did. They have had problems in making a second lot of this antibody and hence still have the same lot that they made few years ago. They are unable to test it in their facility and they have promised to send me a free vial. I am hoping that this will work- else I am in trouble.

[eventhorizon]

Hi again,
The antibody is against human protein produced in Rabbit. I used the same buffer and also a new one. So do you think something in the buffer can selectively block it out? I am getting a new vial tomorrow- so I will try it out with newly made reagents. Hope it works this time

[Anastasia_S]

It seems unlikely but who knows? If that happens to all rabbit Ab you use.. but not the mouse ones, and they are not from the same lot

Its totally unrelated but in our lab an assay stopped working coz the manufacturer changed specifications of the media we used and traces of metal ions started interfering with the procedure (wasnt a WB though).
Maybe if you could borrow some rabbit Ab off another lab and test it with your protocol, it'll rule out the possibility