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western blot problems
Posted by: lilacwine (IP Hidden, New member, 7)
Date: August 9, 2005 09:28AM
I'm testing a new antibody against my protein and finally I had a very good result after a lot of months. The western was perfect, only my band , no background or aspecific bands. I tried again and now I have the same problems:
high background and a lot of aspecific bands around my protein What can I do?? I used the same condition Please help me!!
Re: western blot problems
Posted by: EANON (IP Hidden, Junior member, 19)
Date: August 9, 2005 10:18AM
You used the same conditions? Same antibody concentration, same level of protein, wash, etc?
Perhaps your antibody deteriorated over the course of time. Do you keep it at -80?
Re: western blot problems
Posted by: lilacwine (IP Hidden, New member, 7)
Date: August 10, 2005 02:13AM
yes i used the same condition, the same concentration etc...and the antibody is good....
i stored it at -20° as usual... what can i do to reduce the aspecific bands?? work with the secondary antobody dilution or with the washes?? I don't know....
Re: western blot problems
Posted by: EANON (IP Hidden, Junior member, 19)
Date: August 10, 2005 08:45PM
FYI, you should keep antibodies at -80. The reason for this is that the temperature is not -20 in many parts of the -20 freezer (especially near the door) and proteins are much more stable at -80 than at -20.
That said, there has to be something different about what you did. If you are seeing many nonspecific bands, try decreasing your primary antibody concentration (after blocking for 1 hour). Don't take shortcuts with the washing. Good luck.
Re: western blot problems
Posted by: lilacwine (IP Hidden, New member, 7)
Date: August 11, 2005 03:14AM
thanks so much. I will try.
nice day
Re: western blot problems
Posted by: bing bong (IP Hidden, Unregistered user, )
Date: September 8, 2005 12:00PM
you need to get new antibodies bub
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