(Help) Questions about Western - (Jun/24/2005 )

1. About re-blotting
I just wanna re-develop the membrane with another developing solutions. Ppl in the lab told me (1) block the membrane (2)Incubate with the second Ab and then washing and developing. I am confused whether the 1st Ab are still on the membrane, which had been dry for about 12 h. And why do I need to block the membrane again.


2. About the buffer
We used TTBS. In the first time I used 2*TTBS (300 mM NaCl) for all the
steps, and clear bands appear. Then I realized the mistake and tried 1* TTBS. But the bands are very very weak. Next I used the 2* and nice bands show again. Really confused about the results. Can anybody discuss the influence of ionic strength on the bindng between antigen and antibody? Tks

hi
i would recommend to strip your memebrane and again mae a cycle Block/1st ab/wash/2nd Ab/development. first antibody can remain on the membrane after such time but if so, part of it would be blocked by your first secondary antibody, making interference with your second sencondary antibody procedure.
by the way as you washed your memebrane several times befor development, it's necessary top reblock it to avoid unspecific bindings of your secondary antibody. It would not be as unspecific as if you haven't block your membrane in a first assay but will no be that negligible.

for your buufer conditions : basically the more strenght conditions are, the more specific signal is. But in your case if you get better results in more restrictive conditions i would keep them.

Agree with Fred

you need to strip the membrane and reprobe. I strip it with a mercaptoethanol buffer at 60 C for 25 mins and wash it with a Nacl based buffer 3X 10 mins. Then block with milk and primary..secondary etc.

lemme know if you need the recipe for the stripping and washing buffer.
good luck
p..

hi
there was a discussion on stripping/reprobing membranes here

Thank you so much for ur guys' nice suggestions.

I am sorry for not saying the first question clearly.

In the current lab, we use a regular kit for developing HRP-conjugated 2nd Ab. But if this does not give clear band. We may try a superkit from Phamarcia. Then ppl told me

1. the first Ab is still on the membrane, though the memebrane had been
dry for a while.

2. I need directly block the membrane , then incubate it with 2nd Ab, and so on.

Are the above 2 points right? I am confused about them. Why do I need re-block?


In the lab, ppl do not strip membrane for re-blot with the second 1st Ab, because the PI think stipping would cause much loss of protein. Do your guys think so too?

Thank you again!

hi
actually in order to check this hypothesis, i did do a first analysis, a stripping reprobing to check an other protein, and again stripping and check for the first protein. Results were identical on this first protein. Ok i suppose that you can't strip many times your memebrane but two or three times are still ok.
Fred

PS: i use nitrocelluloses membranes.

Thank you for your guys' info

In the current lab, if I do not get nice bands with the regular developing kits. The PI let me do as following with a superkit

1. Block
2. Incubation with 2nd Ab
3. Developing

I am confused:
1. Is the 1st Ab still on the membrane? And do I need incubate the
memebrane with 1st Ab again.
2. Why do I need to block?

Thank you.

By the way, what is the force for antigen-antibody interaction. electrostatic or hydrophobic?