Western blot of urine samples - Problems verifying loading (Sep/29/2010 )
Hello,
I am running western blots using clinical samples (urine). My problem is that GAPDH and b actin cannot be used as controls to normalise against for loading. Does anyone out there have any experience running westerns on urine? How do you prove you are indeed loading the same amount of protein in each lane?
I was using a strataclean kit which was supposed to be pulling down the same amount of protein in each sample but the gels have extremely high background and it appears that there are varying amounts of protein present in the sample replicates so I cannot trust this method. I read some papers where they load a set amount of the sample (20-30ul) to the wells and I tried this and the gel is much cleaner and the bands much sharper.
The problem is that for my thesis I need to prove the loading is correct so I really need to find a method that allows me to visualise the loadings. I have tried to use Ponceau but even after an hour there is no staining. I know the ponceau is working because it stains membranes with protein from cells. I also know I have successfully transferred protein as I get bands in my westerns after probing with antibodies.
I know I could do commassie staining but then I cannot transfer the proteins from a gel to the membrane for probing with antibodies. It has been suggested to run duplicate gels, transfering one and staining the other but I don't think this would be acceptable if I wa to try and publish it.
Has anyone any suggestions how to stain for proteins and then transfer?
Thanks in advance for any suggestions!!!!!
siscady on Wed Sep 29 14:20:28 2010 said:
Hello,
I am running western blots using clinical samples (urine). My problem is that GAPDH and b actin cannot be used as controls to normalise against for loading. Does anyone out there have any experience running westerns on urine? How do you prove you are indeed loading the same amount of protein in each lane?
I was using a strataclean kit which was supposed to be pulling down the same amount of protein in each sample but the gels have extremely high background and it appears that there are varying amounts of protein present in the sample replicates so I cannot trust this method. I read some papers where they load a set amount of the sample (20-30ul) to the wells and I tried this and the gel is much cleaner and the bands much sharper.
The problem is that for my thesis I need to prove the loading is correct so I really need to find a method that allows me to visualise the loadings. I have tried to use Ponceau but even after an hour there is no staining. I know the ponceau is working because it stains membranes with protein from cells. I also know I have successfully transferred protein as I get bands in my westerns after probing with antibodies.
I know I could do commassie staining but then I cannot transfer the proteins from a gel to the membrane for probing with antibodies. It has been suggested to run duplicate gels, transfering one and staining the other but I don't think this would be acceptable if I wa to try and publish it.
Has anyone any suggestions how to stain for proteins and then transfer?
Thanks in advance for any suggestions!!!!!
Hola, I never have worked with urine samples, but there is low protein in them if there isnŽt any pathology. I would estimate the concentration os protein by Bradford and IŽll try to put at least 5-10 ug /well to have any stain by Ponceau. If the volume necessary were too high I would made a TCA precipitation. Good luck