Problems in Western blot ! - need help for my western blot (Jun/08/2005 )

The result of my western blot is never stable. Sometimes it works fine, sometimes it don't. And I don't know why! My research is concerned about the protein trafficing. So I have to test for several proteins by western. For cargo protein, my antibodies worked fine. For carrier protein, which has 4 subunits (two big, two small). The strange thing happened. For example, when I use different primary anitibodies, then use same secondary antibodies. The banding patterns are always the same. Even I tried a different secondary antibody for one of the subunit, it also shows the same banding pattern. Does anyone have the similar problem?

Another problem is my Molecular marker. We bought chemluminescent marker from Pierce. It never works properly. Can you recommend me a similar product?

Thanks in advance for your help.

try mol markers from neomarkers..
p..

Thanks for your response. Do they offer chemiluminescent marker?

Hi, I don't get so well your problem. Are u looking for the different subunits in your gel?
If this is it, maybe you are having the same problem as me with subunits not being properly disassembked in the gel maybe because of low concentration of ME or DTT. I was advised here, to work with increasing amounts of reducing agent, and see what happens.
If I got it wrong, maybe you can explain it better.

About chemiluminiscence, I use it but I run a prestained marker, that once transferred I cut away and keep sealed in plastic. It works fine for me. Just over expose to see the borders of your membrane at the end to be sure the correlation with your marker.

Good luck!

M

Thanks for your suggestions. Yes I am looking for the four subunits of a big heterotetrameric protein complex. The two big subunits are 120 and 130kD. And the two small ones are 26 and 47kD. I used 5% β-mercaptoethanol in my sample buffer instead of DTT. I will try to increase the reducing agent.
Thanks again for your kind help. One more question, which compnay do you purchase your chemiluminescence marker? My marker was also prestained, but it never show all the bands, only the first four bands showing all the time.

Another problem is the primary antibodies I used are not specific to the species I studies. Will that be a reason for my failure?
Thanks.