dot blot and western blot

[Liang]

Dear All
I am so glad to find this great forum and be a member of it!
I've tried western blot and dot blot for ~2 months. I used mouse mono and rabbit polyclonal antibodies and Qdot conjugated 2nd antibodies. Protocol and blocking reagent were from GE Healthcare ECL Plex (but I did not use their secondary antibody).
I did get much brighter signal using QDots conjugated antibodies than the Cy3, 5 conjugated dyes with dot blot. However, sometimes I did not have signal or the whole membrane was completely black (full of signal). I do not know why.
When I tried western blot using the same immunostaining protocol but transferred from SDS-PAGE after separating the cell lysate (purchased from ProSci), I could not see any band. I am pretty sure the transfer was OK as I see the protein marker on the membrane and I did the transfer (using iBlot from Invitrogen) hundreds of times, never having any problems).
Can anybody help why I do not the signal from the western blot? Why my results with dot blot were not very reproducible?
Here is my brief protocol:
1. Block the membrane in 2% ECL Advance blocking agent in wash buffer, shaking at 4˚C over night or at room temperature for 1 hour.
2. After blocking, rinse the membrane twice in PBS-T and then wash 2 x 5 minutes.
3. Incubate the blocked membrane (protein side up) with primary antibodies, respectively, for 1.5 hours shaking at room temperature.
4. Rinse the membrane twice in wash buffer, then wash the membrane for 2 x 5 minutes in wash buffer at room temperature, shaking.
5. Dilute Goat anti-Mouse-Qdot 655 (Q11022MP, Invitrogen) (1:1000) in blocking buffer.
6. Incubate the corresponding washed membrane protected from light for 1 hour at room temperature, shaking.
7. Rinse the membrane three times in wash buffer, followed by 4 x 5 minutes in the same buffer shaking at room temperature and protected from light.
8. Rinse the membrane three times in wash buffer (without Tween 20).
9. Detect the signal by scanning the membrane using Typhoon.

Thank you in advance!

Liang

[Liang]

Sorry there was an error. I dilute 2nd antibody in PBS, not in blocking buffer. In fact, I could not get any signal if 3nd antibody in blocking buffer. Plus, signal to noise ratio was significantly reduced if 2nd antibody was diluted in PBST.
Thanks!