Hi all,
Recently I have been having trouble exposing my tubulin. There seems to be an uneven exposure of the lanes, yet my ponceau looks even across. From what I have read online, this could possibly be due to uneven antibody exposure, however I use the same method for all my antibodies and am only having trouble with tubulin.
My methods:
1. After transfer, add blocking buffer (depending on antibody: %5 milk or 5% BSA) for 1 hr
2. Prepare primary antibody solution at 1:1000 in 1x TBS-T. Incubate membrane overnight on roller.
3. Wash three times for 5min each (total 15min) with 1x TBS-T.
4. Prepare secondary antibody at 1:2000 in 1x TBS-T. Incubate 4C for two hours on roller.
5. Wash three times for 5min each (total 15min) with 1x TBS-T
6. Prepare ECL solution at 1:1. Incubate membrane for 1min each and then expose using film/developer
My troubleshooting so far:
1. Buy new non-fat milk (ours was expired)
2. Decrease/increase antibody concentrations
3. Apply ECL directly to membrane and expose immediately (to avoid signal being lost quickly)
4. Prepare fresh antibody every time
Any tips would be helpful. Attached is a picture for reference.
Hello Anna P Stephan
There is a possibility that when used at a higher concentration, some antibodies exhibit a tendency to form aggregates that reduce its binding capacity. These aggregates can be removed by centrifuging the antibody vial at high speed for 5-10 minutes followed by careful pipetting out of the antibody solution (supernatant) without touching the bottom of the vial.
The protein aggregation process is driven by several factors, which includes amino acid composition and sequence, environmental factors such as pH, concentration, buffers/excipients and shear-forces during processes used for protein production, as well as final formulation and storage conditions.
I feel 1:2000 for secondary antibody is too high a concentration. For Western Blot, use a concentration in the range of 1:5000 – 1:30000 for the secondary. Also, make sure that the membrane is covered evenly with the antibody solution, and do not allow the membrane to dry.
I have also noticed that you incubate the membrane with secondary antibody for 2 hours at 4 degree C. You should incubate the membrane with secondary for 1-2 hours at room temperature and not at 4 deg C.
Hope these tips help!
Best.
Agree with Malcolm Nobre , Whenever we use secondary antibody the concentration is around 1:10000-1:20000. And I also recommend incubating for 1-2h at RT for the secondary antibody.
Also when you apply the ECL I would use saran wrap to pipet some on (like 500ul of Sol A. + 500ul of Sol B.) and then flip the membrane facing the front where the protein is attached, down on top of the pipetted ECL.
This way you expose the membrane to the liquid at the same time no matter what. Do be carefull with airbubbles, you have to remove those by pressing on the membrane with some tweezers.
Lastly I wanted to comment how cool it is that some people still do this with film and a developer. I only did this during my first internship but there was something really cool about going into the dark room and seeing the results that way. Good luck!
Malcolm Nobre Koen van Wijk thank you both for your responses! I did try reducing the secondary antibody as well as placing the membranes on a shaker instead of in tubes on a roller. I have had luck so far with these methods but I will also try my secondary at room temperature.
Koen, thank you for the tip about the ECL, I have seen others do this but have never tried so I will give it a shot. This is the first lab I have been in that uses a developer, but I enjoy the old school!