Hi Paul, You should perform everything in a cold room, making sure you don't let the samples heat up (even room temperature can externally uncouple eNOS). Don't boil the samples but keep them cold after adding sample loading buffer. Load the gels in the cold room. Run on approximately 50V for 4-5 hours in the cold room, place an ice pack in the buffer tank. Transfer at about 75V for 2 hours, also cold, on a magnetic stirring plate. Block with 5% milk. Santa Cruz enos antibody works fine. You need a femto kit to get the signal. This should work. Good luck!

thanks for the suggestion. i follow cold conditions during my electrophoresis run. but i do my transfer at room temperature. I can easily trace monomer, while the dimer form of eNOS is inconsistent. What lysis buffer have you used.

RIPA lysis buffer. Perhaps you should try to perform transfer in the cold room also (with ice packs).

I have even tried cold transfer. I get a smear, instead of intact band

What tissue do you use? When Laemmli is kept cold, it congeals sometimes. I homogenize very briefly before I load the sample. The problem must be in the tissue itself. If you keep enough protein content, you can think about centrifuging your sample right before adding laemmli.

I am using endothelial cells. if i subject the lysate to sonication, it leads to monomerization.

If i do not sonicate it results in protein accumulation in well, as it does not resolve.

If i use REPA buffer containing SLS, it monomerizes.

Hope you have also observed these with your tissue samples.

Your suggestions would be appreciated.

Hi Christa and Paul, I managed to tweak the protocol. I have used a small mixer to homogenize my tissue. When I stopped doing that and cut the tissue very small with a scalpel, the monomers disappeared from the blot. Other important stuff is not to use mercapto ethanol and perform the electrophoresis on ice. Normally, it should work.

Cheers

Jan

I had a similar problem with proteins 'congealing' in the wells after mixing tissue homogenate with cold laemmli. I let it shake for 10 minutes in a cold room (with RIPA) and then centrifuge them at 9000 RPM for 5 minutes.

Hi Christa, I'm working on a paper for eNOS but I'm not ready with my experiments yet.I based my experiments on a paper by Dong et al. but I don't have the link at my hands now. You must NOT homogenize at all. Just let the tissue soak in the laemmli after making carpaccio of the tissue and do NOT homogenize, just let it shake for 10 mins. Just centrifuge in the cold room at 4°C, take the supernatant and add laemmli to it. Good luck!

Jan

Hi Crista,

I use endothelial cells in my experiments and not the tissue. However the technicality would not differ. I used Lamelli buffer by Bio-Rad and did not use b-mecraptoethanol. The experiment should be run under cold condition. I place the blotting apparatus in a ice bath and run a native PAGE at 40V for 6 hours. I recommend to use narrower wells and use 20-30ug protein.

 Good luck

Hi paul and Christa

Did you find solutions for your problems because i will do the same know and i have no exprience in that work, i am working with endothelial cells and i repeated western for 6 times with no result at all. I want to ask another question should i make purification for eNOS first then run purified enzyme on gel or i can load the whole lysate from cells and to get dimer band how can i differentiate between monomer and dimer band?

Thanks in advence