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!