Adrenergic receptor proteins (western blot problems) - (May/10/2007 )

Hi everyone,


I have a problem with western blotting of alpha 1 A, B and D adrenergic receptors proteins in rat brain (prefrontal cortex and hypothalamus) (.
I use Santa Cruz prim. and secondary antibodies and the problem is that I can not see the bands of my interest. I am afraid that I just don't extract those membrane proteins properly
I tried many homogenization buffers (RIPA, SDS 2%, hot SDS 2%) but I can't centrifuge the supernatant at 100 000xg ( 28000xg is the maximum which I can obtain).

I got some weak results when homogenized tissue in 1 % SDS and 10mM tris-HCL (with protease inhibitors) with polytron homogenizer. I centrifuge homogenates at 28 000xg and resuspend pellet in hom. buffer. But when I try to repeat western from the same sample there are no bands on the membrane (.

Anyone knows anything about those proteins??

Thanks,

Hi everyone,


I have a problem with western blotting of alpha 1 A, B and D adrenergic receptors proteins in rat brain (prefrontal cortex and hypothalamus) (.
I use Santa Cruz prim. and secondary antibodies and the problem is that I can not see the bands of my interest. I am afraid that I just don't extract those membrane proteins properly
I tried many homogenization buffers (RIPA, SDS 2%, hot SDS 2%) but I can't centrifuge the supernatant at 100 000xg ( 28000xg is the maximum which I can obtain).

I got some weak results when homogenized tissue in 1 % SDS and 10mM tris-HCL (with protease inhibitors) with polytron homogenizer. I centrifuge homogenates at 28 000xg and resuspend pellet in hom. buffer. But when I try to repeat western from the same sample there are no bands on the membrane (.

Anyone knows anything about those proteins??

Thanks,

QUOTE (silver80 @ May 10 2007, 05:26 PM)

Hi everyone,


I have a problem with western blotting of alpha 1 A, B and D adrenergic receptors proteins in rat brain (prefrontal cortex and hypothalamus) (.
I use Santa Cruz prim. and secondary antibodies and the problem is that I can not see the bands of my interest. I am afraid that I just don't extract those membrane proteins properly
I tried many homogenization buffers (RIPA, SDS 2%, hot SDS 2%) but I can't centrifuge the supernatant at 100 000xg ( 28000xg is the maximum which I can obtain).

I got some weak results when homogenized tissue in 1 % SDS and 10mM tris-HCL (with protease inhibitors) with polytron homogenizer. I centrifuge homogenates at 28 000xg and resuspend pellet in hom. buffer. But when I try to repeat western from the same sample there are no bands on the membrane (.

Anyone knows anything about those proteins??

Thanks,

Are you sure your antibodies work? Has anyone in your lab used those before and got good results? Maybe this sound bad but I am a bit skeptical about Santa Cruz Antibodies (some from them do work well, but others just plain bad)

I have never worked with this receptor, so here is just suggestions. Ok, first, if they use other tissue and got good results, maybe you can try for a positive control from the same type of tissue or cell lines (any cell line has this or only tissue???) and see if you can get the same result?

Second, if you think that it is because they use higher g force, then your protein should be in the supernatant still (you are checking the pellet, right?). How about check the sup. or maybe total lysate? If you can find them there, then it is because you did not extract the protein properly. How come you can't use other centrifuge anyway? If you can't find the band even in the total lysate, something wrong with WB.

Third, if you are sure others use the same Ab and got good results, my sincere appologize to Santa Cruz. You will have to play with the condition of your WB then, Ab concentration, time of blocking, probing, washing, exposing...

Thanks for reply

I also try with supernatant ( I always took sup. and pellet -just in case) but with no result. I think that the problem is that those subtypes of receptor don't abound in my tissues ( not like in hear or arterial vessels). I found an article where whole brain had beed taken or celebral cortex, in my lab we do dissection and research only some of the brain structures . I tried to do longer transfer (with standard transfer buffer-membrane looks ok after ponceau S staining). I also block membrane overnight ,than 1 hour-2 hours , and probed overnight with prim. ab. But I found the lab where I can centrifufe at 100 000g ,and hope that it would help. If not , maybe those antibodies are just not suitable for my western( cos I also read something bad about them).

I don't see how centrifugation at 100 000g affects the presence of your protein...

Do you add Triton and/or NP-40 to your lisate? Maybe the receptors are caught in the membrane and are therefore not "detectable".

It is true thought that maybe these receptors are not present in the brain... Also, as you said, maybe these antibody are not good for Westerns.

Anyway, good luck!

Hi Madrius,

Thanks, I want to try this centriugation because I've found that step in most protocols concerning adrenergic protein. I used triton-X 100 and also NP-40 but with the same "blank" result :/. But tomorrow I have another trial:))

First make sure your Abs all work well. After that, try extract proteins etc... But I really think you need a positive control for WB first (heart or arterial vessels or whatever), make that work before going to do more with brain.

Hi,

I wish I could try with positive control. But I don't have enough time to do it , I'm stayining in my lab only two weeks more :/ , I will just leave notes and directives for someone else. Many thanks for helping me

First things first... As many people have pointed out, make sure the antibodies work. The easiest way of doing this is to not buy them from SC! Check the literature and see if anyone have published anything with the antibodies, then they are likely to be good! I spent almost a year in trying to get SC antibodies to work for beta adrenergic receptors. Also, try using both nitrocellulose and PVDF membranes, could make a huge difference for some reason.

D