Look for your antibody, check with dot blot techniques,
Perhaps you incubate with primary antibody little longer, wash properly and wash with BSA copiously and the
n u add secong conjugated antibody, make sure it properly binds and wash more times. If you get the entire thing gets labeled , it means your anti body is bad or second conjugated is bad. You have to check it out.
Raj
--- On Sat, 21/11/09, kantharaj gowda <grkraj_blr@yahoo.
From: kantharaj gowda <grkraj_blr@yahoo.
Subject: Re: [M.B.N] western blot with serum
To: molecular-biology-
Date: Saturday, 21 November, 2009, 7:01 PM
Hi
Aisf Raja.
Are you the student of Jains college or what?. Thinking that you the student of mine i am telling you. If you have antibody, you should know maximum point in antigen and antibody titration. If you don't know you have to take a chance and do it with different concentration of IgGs; or ask who has provided that antibody.
After blotting and incubating with serum, you have wash in equillibrium buffer to remove the serum many times and then add your conjugated antibody and follow the protocol. Where are you working? it take ore than 12 hrs; do you have a walk-in cold room?
Raj
--- On Tue, 17/11/09, Asif raza <asif1308@yahoo. co.in> wrote:
From: Asif raza <asif1308@yahoo. co.in>
Subject: Re: [M.B.N] western blot with serum
To: molecular-biology- notebook@ yahoogroups. com
Date: Tuesday, 17 November, 2009, 3:23 PM
to me its the concentration of antibody which you have to optimized. Run ur blot with different conc. of 1st antiobody.
--- On Tue, 17/11/09, Zahra Moradpour <moradpour_z@ yahoo.com> wrote:
From: Zahra Moradpour <moradpour_z@ yahoo.com>
Subject: Re: [M.B.N] western blot with serum
To: molecular-biology- notebook@ yahoogroups. com
Date: Tuesday, 17 November, 2009, 1:23 AM
Hi
I just took a glance to your steps and I could not find the blocking step before adding the first antibody, the washing of secondary antibody is not enough, its better to wash 4 times in 1- 2 hours, you need to search for protocols online especially the ones from companies web sites or in books like sambrook molecular cloning a laboratory manual.
good luck
--- On Mon, 11/16/09, caterinaiofrida <caterina.iofrida@ bioclinica. unipi.it> wrote:
From: caterinaiofrida <caterina.iofrida@ bioclinica. unipi.it>
Subject: [M.B.N] western blot with serum
To: molecular-biology- notebook@ yahoogroups. com
Date: Monday, November 16, 2009, 4:51 PM
Hello,
I am using western blot to detect antibodies in human serum. I have a purified protein (cochlin) which I transfer on nitrocellulose membrane (after SDS-page electrophoresis) . I manually cut the membrane in strips after transfer, I put the strips in an immuno tray (Roth) for incubation with serum diluted in blocking buffer, then I wash 3 times (10 minutes each) in TBST in larger boxes, I put secondary antibody (using immuno-tray again), I wash 2 more times in TBST and 1 in TBS, then I use a chemiluminescent substrate and develop on film. For every strip I put 2 ml of every solution for incubation.
My problem is that often I have signal on the whole strip, so I think that incubation steps are not efficient in the tray the way I use it or there are some other problems. Do you have any idea about it and maybe an advice to give?
Thank you very much
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