Hi @Stanislav N Naryzhny
I recently tested your method "Blue Dry Western" with some antibodies that were not resulting in good clean bands by the classical western method. Unfortunately, the only bands I got with your method were with the anti b-Actin loading control antibodies. With the other targets I only got either a very faint band or no bands at all. I loaded 20 ug of protein from cell culture lysates.
I imagine that one of the problems is that my targets are not as abundant as b-Actin, so that might diminish the ability of the antibody to detect them in the membrane.
Do you think I could solve this problem by incubating the dry membrane for a long period of time, like 1-2 hours at RT? Or maybe adding 0.1-0.5% Tween20 might improve the antibody's ability to reach the target?
How about reducing the pore size of the PVDF membranes (from 0.45 to 0.2 um) and diminishing the time of protein transfer from the gel to the membrane, to make sure most proteins I transferred remain superficial in the membrane without penetrating too much in the membrane?
Let me know if you have any ideas about how to detect low abundance protein with your method. I really would love to be able to use your method for all my westerns.
Thanks a lot
Hi Yoskaly,
There are some ideas how improve the sensitivity of your antigen detection.
1. Load more than just 20 ug of the lysate. Try 40 ug, may be more (check only not to overload the well).
2. Increase concentration of your Abs (if you are using 1:1000 then make 1:500, or even 1:250). Sure your Abs are expensive, so you can make the minimal amount of their solutions to save them. You can make only 200-500 ul of Ab solution just enough to cover the area on the membrane, where your antigen should be located (as the membrane is stained you can easily determine it). Spread it around this area by the pipet tip and leave on the bench for 30 min (same time for primary and secondary Ab). As you see, in this case you will not need to put the membrane into the bag and rotate it on the rotation platform! As we checked, there is no need of rotation during Abs incubation!
3. Don't increase the time of incubation more than 30 min and absolutely don't add Tween20!!! It will only make the background dark!
4. I didn't compare membranes with 0.45 or 0.20 pore size, so can't say that 0.20 will work better. My guess - not
5. Don't decrease much the time of transfer. May be you even need to increase it! Be sure that your antigen transferred from the gel to the membrane! As it's minor you need to transfer it in sufficient amount to be detected by Ab. Just stain the gel after the transfer to see the efficiency of the transfer. Actin is very abundant protein and to be detected on the membrane you need to transfer onto membrane only small portion of it.
Hope it will help
Regard
Stanislav Naryzhny
Thanks for your suggestions!
-The first thing I will try is your suggestion of increasing the concentration of antibody and the incubation of a small amount of ab. without shaking.
-Normally I assess the quality of the transfer by the intensity of the colors of the protein markers transferred to the membrane. it is usually good. But I will stain the gels to double check.
-I really want to avoid increasing the protein loading of my samples because I only have a limited amount for each experiment and I have many westerns to do with them. So I will try that last.
-Regarding the idea of adding a small concentration of tween 20. Well, I got it from the paper by Mansfield ( Article Rapid Immunodetection on Polyvinylidene Fluoride Membrane Bl...
) saying that "Tween 20 at 0.05% was necessary for the best sensitivity". So I was wondering if there is a sweet spot of Tween concentration that can be used for optimal sensitivity/background ratio.... I guess you might disagree with that.Anyway, thanks a lot and I will let you know about my results!
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology