Western Blot troubleshooting: band is too high - LiCor imaging of Alpha tubulin membranes (Nov/01/2005 )
Hello everyone...I'm new here, so try and bear with me.
Unlike all you skilled veterans out there, we use pre-packaged gels (4-12% bis-tris with MOPS running buffer) from invitrogen, i'm not sure if anyone else has had any experience with them, but I also use their NuPage LDS sample buffer to extract my samples. I've used both chemiluminescence and infrared imaging (Odyssey LiCor) to visualize my membranes. With both methods, the bands produced (staining with monoclonal alpha-tubulin sigma, DM1A) are way to high for alpha tubulin (which should be around 55 kDa), I am getting bands at 100. The first time I tested out this Ab it gave me the right location, however on subsequent runs, it has been high. Is there any reason that it should be dimerizing? Is there something that is cross reacting?
Thanks for your help!
What are you doing to the protein?
In theory if you are doing the same thing every time then the band SHOULD be in the same place, of course theory and practice are two totally different things. Are you denaturing your protein for long enough? That is the only thing I can think of that would cause it to run poorly. Also is your marker running at the same speed as your samples? If the marker is shooting out the bottom of the gel at high speed while the samples are running normally then that could cause an apparently wrong result, however I'm not sure how that would happen.
Rosie
Do you have reducing agent in your sample buffer ? Sometimes you can see dimers of proteins
2x55= 110 so it might be what you see in your assay
Pesji 
Unlike all you skilled veterans out there, we use pre-packaged gels (4-12% bis-tris with MOPS running buffer) from invitrogen, i'm not sure if anyone else has had any experience with them, but I also use their NuPage LDS sample buffer to extract my samples. I've used both chemiluminescence and infrared imaging (Odyssey LiCor) to visualize my membranes. With both methods, the bands produced (staining with monoclonal alpha-tubulin sigma, DM1A) are way to high for alpha tubulin (which should be around 55 kDa), I am getting bands at 100. The first time I tested out this Ab it gave me the right location, however on subsequent runs, it has been high. Is there any reason that it should be dimerizing? Is there something that is cross reacting?
Thanks for your help!
I think you should add DTT, final concentration: 100mM
Hi,
I use the invitrogen 10% gels and have also a band in the wrong size (always about 55kDA). It shows up with two different antibodys.
Have not found the problem yet... :-)
superawo
It's either a dimer which should dissociate if you add reducing agent to your sample buffer, or severe glycosylation.
Unlike all you skilled veterans out there, we use pre-packaged gels (4-12% bis-tris with MOPS running buffer) from invitrogen, i'm not sure if anyone else has had any experience with them, but I also use their NuPage LDS sample buffer to extract my samples. I've used both chemiluminescence and infrared imaging (Odyssey LiCor) to visualize my membranes. With both methods, the bands produced (staining with monoclonal alpha-tubulin sigma, DM1A) are way to high for alpha tubulin (which should be around 55 kDa), I am getting bands at 100. The first time I tested out this Ab it gave me the right location, however on subsequent runs, it has been high. Is there any reason that it should be dimerizing? Is there something that is cross reacting?
Thanks for your help!
Similarly:
Does mercaptoethanol or DTT as a reducing agent present in NuPage LDS sample buffer? It should be added just before use 
I also met with that problem for some time before, because this important infromation was missing in a local protocol !