It was not possible to see any expected band for the target, with a very low molecular weight (8-12 kD), by WB. The membrane was very clean after incubation with Ab-I-AbII. Probably we lost the protein during the electrophoresis because of its size. Then we decided to use Dot Blot and we got very nice dots for that Ab-I. We know that Dot Blot is a simple and quick assay that may be employed to determine if antibodies and detection system are effective and to determine appropriate starting concentration of primary antibody for western blot.
But with clean strips for a certain Ab (so, no aspecific bands), Would Dot Blot be of help when WB does not work to get information/indication of that target after drug treatment? If so, How is it possible to argue this problem?do you have references about Dot Blot please?
Thank you in advance
Best Regards
Mario
Dear Mario,
Please use two membranes during transfer of low molecular weight proteins. If they pass through first, chances are they'll get captured on the 2nd membrane underneath the 1st one. You can also try to optimize transfer conditions (voltage, time etc.) to ensure that your low molecular weight proteins are not passing through. I do not think dot blot can be used in place of Western blot for obvious reasons.
1) Yes, the Dot Blot can be helpful for identification of the target when WB does not work for some antibodies / antigens.
2) When you use your sample for SDS-PAGE in Laemmli buffer you usually use it under reduced conditions in the presence of SDS. When you use your sample for Dot Blot (DB) you usually load your sample on the membrane without b-ME or SDS. That means you load your sample in the non-denatured conditions. So, your particular antibody will have more chances to recognize your protein.
3) When you use Dot Blot you have much higher concentration of the antigen loaded ~ 10-100 fold higher compared to your WB amount (relative to the area) loaded for your WB.
4) Yes, "Dot Blot is a simple and quick assay that may be employed to determine if antibodies and detection system are effective and to determine appropriate starting concentration of primary antibody for western blot".
5) You need to remember, that the antibody recognition of your protein target depends on the conformation of the protein (reduced or non-reduced) and that could make a big difference between WB and DB results.
6) Usually, DB is used when it's necessary to screen multiple samples / protein antigens with the one particular Ab to figure out the initial conditions of the Ab concentrations (or antigen amount), or salt concentrations, or just the pH of the buffer for the interaction.
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