Need help! Problems on Western blotting - (Oct/29/2006 )

Can anyone tell me the reason why I cannot get any image using Chemiluminscence? I have tried once before and the signal is very strong. But then, up till now, no signal or even background can be seen under the LumiImager..Whatz happening?
My procedure is : Blocking-->Washing__>1st antibodies-->Washing-->2nd antibodies-->washing-->add substrate-->Chemiluminscence
Is there anything wrong in my procedure?
Also, If I want to try the western blotting once more using the same membrane, what chemicals can I use to wash off the attached antibodies?

stripping buffers :
0.2M glycine ph 2.5 / tween 0.05% for 30' at 80°

or

B mercapto ethanol 100mM (104µl from 14.3M stock)
tris 62.5mM ph 6.8 (6.25µl from 1M stock)
SDS 2% (20mll from 10%stock)

56° for 30'

hi
I don´t see anything wrong in your protocol.
Have your checked your transfer? for example with a Ponceau staining of your membrane or by using prestained molecular weight markers. In my opinion, first of all you have to be sure that your transfer it´s ok and so you have proteins on your membrane.

Have you tried increasing the exposure time?
Is your stuff in good conditions?
Are you sure you are working with the right side of the membrane? I mean, the side which has the proteins. This hapenned to me once

Here there are advices for stripping:

stripping1

stripping2

hope this helps

Thank you for your advice. I am sure that I have the right side of membrane and the dye markers are clearly trasferred to the membrane. Thank you for the stripping method.

hi
I don´t see anything wrong in your protocol.
Have your checked your transfer? for example with a Ponceau staining of your membrane or by using prestained molecular weight markers. In my opinion, first of all you have to be sure that your transfer it´s ok and so you have proteins on your membrane.

Have you tried increasing the exposure time?
Is your stuff in good conditions?
Are you sure you are working with the right side of the membrane? I mean, the side which has the proteins. This hapenned to me once

Here there are advices for stripping:

stripping1

stripping2

hope this helps

That happened to me a few times, everything seems to be ok but there´s no signal and no explanation
Are you sure the working dilutions of your antibodies are right?
Don´t get discouraged Just try again and see what happens.
good luck


[quote name='HenryTcby' date='Oct 29 2006, 06:25 PM' post='74907']
Thank you for your advice. I am sure that I have the right side of membrane and the dye markers are clearly trasferred to the membrane. Thank you for the stripping method.

I can't see any wrong in your procedures.

Is your ECL fresh or are the solutions perhaps too old? How long do you incubate in your ECL? How long do you expose your membranes? How long do you block and in what blocking solution?

Hi were u able to slove the problem. I am having th esame problem with me westerns. dont see any signal at all. I have tried lots of thigs but nothing seems to work.