Western Crisis: Sudden Disappearance of a Signal - (Sep/14/2008 )

Hello. This post is long, but I've tried to be thorough, so please read, because this undergrad desperately needs your help.

I've been trying to solidify a protocol for a western blot for a 180kD mouse protein from skeletal muscle homogenate using two different primary antibodies: one mouse monoclonal and one goat polyclonal. I first used the polyclonal some months ago and got a fairly strong signal using an Amersham Bioscience ECL kit, with lots of non-specific binding, little background noise. This told me my technique was good, but the antibody was bad for this purpose, so I got a new one, the monoclonal mouse, but kept the goat polyclonal. I also checked the company website's antibody page, and verified that 1) it's known to work for mouse protein, and 2) that they got bands using human cell culture lysates.

With this new primary monoclonal mouse antibody I managed to get plenty of background noise, but no signal at a 1:1000 dilution. I ran a blot on which I used a 1:100 just to establish a signal, hopeful, and I got nothing. Dejection. I re-ran the SDS-PAGE with new protein and had a good nearly complete transfer - nothing. Not even background noise, like I had just ran the film into the X-Omat right out of the box. I'm using the same secondary antibody with which I got background noise. Before this I had already gone through several useless blots due to someone telling me to use an incorrect Alexa-fluor secondary antibody, so I only have about two more shots of the this monoclonal Ab to see if it works and I can tell that my PI is reluctantly willing to purchase one more aliquot of some antibody. I wanted to test my technique before I touched anymore of this after the signal suddenly failed, so...

I return to the goat polyclonal using a donkey anti-goat secondary HRP-conjugated Ab that is of an unknown age (I'm an undergraduate who came into this lab about a year ago and no one labels anything). No signal, no background noise, no nothing. In the development room I can see glowing substrate on the discarded saran wrap on which I prepare the blots, but when I develop, nothing, even after exposing overnight starting about 8 minutes after I've exposed the membrane to the substrate. The film is also of an unknown age, but I used it on other blots and it works. The ECL kit works because 1) I saw the glowing substrate, and 2) a graduate student in the lab had a successful blot using it. On the twin membrane I've stained with Amido Black and see lots of banding, even in the higher molecular weight portion where my target protein resides. That this blot failed using a primary antibody that was worked in the past tells me that something is probably failing in the technique or the reagents sometime after the transfer, and most likely not in the primary antibody. I haven't changed anything radically besides wash times or whether I use Tween in the washes. The biggest difference is that I've completely lost the signal.

One nagging question I have is, Is there a difference between Peroxidase-conjugated and Horse radish peroxidase-conjugated secondary antibodies? Do they both work with ECL detection? Some of the secondary Abs I have say peroxidase-conjugated, while others say HRP-conjugated.

Specs:

SDS-PAGE for 4 hours at 70V in a 4/6% discontinuous gel
reducing protein using DTT
6% SDS treatment buffer
Wet transfer for 2 hours at 85V
PVDF membrane
PBS or PBS-Tween washes (0.01%) (I've used both in an effort to troubleshoot this; same result)
5% fat-free milk, no tween during incubations
overnight blocking at 4 degrees C
2 hours room T primary
1.5 hours room T secondary
~30mins total of washing in between incubations

I would appreciate any help determining where this protocol is failing, or what material may need replacing. Thanks.

Are sure that the developer solution is still allright? Check its colour and make sure that it is honey-coloured and clear, not dark and cloudy. This is essential for getting ANY signal. Try and test a film strip for this by putting something on it (your hand) and then put the light on. When you develop it, you should have plenty of dark signals where you hand had not been. If this is not working, surely something is wrong on the point of developing or with the films.