Western blot problems - Please need Help!!! (Jul/10/2007 )
QUOTE (jyothsna @ Jul 20 2007, 03:08 AM)
Hi there,
I have similar problems with westerns at the moment. to get rid of the background and other non-specific bands I was thinking of pre-incubating the Primary Ab diluted in milk with whole cell extract from a strain deleted for the protein I am probing. Has anyone tried this? and do you have a clue as to how much protein I should be adding to my dilutions? I have also tried blocking with 7% skimmed milk in TBS-T but my westerns after developing came out blank
any suggestions?
Thanks
I have similar problems with westerns at the moment. to get rid of the background and other non-specific bands I was thinking of pre-incubating the Primary Ab diluted in milk with whole cell extract from a strain deleted for the protein I am probing. Has anyone tried this? and do you have a clue as to how much protein I should be adding to my dilutions? I have also tried blocking with 7% skimmed milk in TBS-T but my westerns after developing came out blank
any suggestions?Thanks
check if blotting procedure worked, maybe this could be the underlying cause (amidoblack staining)!
-moljul-
check if blotting procedure worked, maybe this could be the underlying cause (amidoblack staining)!
[/quote]
Yea it did and well, i did a ponceau staining though
-jyothsna-
QUOTE (medchemgirl @ Jul 10 2007, 10:29 PM)
I am having problem with my western blots. I'm a starter in this, and haven't been able to figure out how to get a good WB.
Here I send a pic of a WB. Can anyone have an idea on what could be the cause of those of all of that non specific binding I see in my gel? I can even see the happy face at the bottom of the film which is from the blue line at the bottom of the gel after you run it. And I don't understand all the spotting at the top of the gel, like if part of my protein was degraded but the antibody recognized it. I don't know. I'm clueless.
1ry ab (anti-ERa) = 1:500 for 1:30hr
1ry ab (anti-GAPDH)= 1:100,000 for 30min
2ry ab(anti-mouse)= 1:10,000 for 30min
I blocked with BSA 5% overnight in the cold room, for every wash I did 1, 2, 3, 5, 10 min
I'm going crazy, could it be my samples?? I store them in the -20
I'll appreciate any help
Here I send a pic of a WB. Can anyone have an idea on what could be the cause of those of all of that non specific binding I see in my gel? I can even see the happy face at the bottom of the film which is from the blue line at the bottom of the gel after you run it. And I don't understand all the spotting at the top of the gel, like if part of my protein was degraded but the antibody recognized it. I don't know. I'm clueless.
1ry ab (anti-ERa) = 1:500 for 1:30hr
1ry ab (anti-GAPDH)= 1:100,000 for 30min
2ry ab(anti-mouse)= 1:10,000 for 30min
I blocked with BSA 5% overnight in the cold room, for every wash I did 1, 2, 3, 5, 10 min
I'm going crazy, could it be my samples?? I store them in the -20
I'll appreciate any help
Is this a non-purified antibody? have you tried purifying it to get rid of all the non-specifics?
-smoochiepie79-