Dear Scientists
After protein expression, I did periplasmic and total protein extractions. I did the dot blot test to see if my proteins have been expressed. well, The dot blot result was perfect and I saw the brown dots. but after this I did western blot and I saw no bands on my membrane. Has anyone faced the same problem? what can go wrong to cause this problem?
Do you have all the required negaive controls in your dot blot to be sure that your positive result is true? You could use as negative controls a protein extract from the same cells you are using to express the protein (without the recombinant protein, of course) and also an unrelated detection antibody to reveal the dot blot in parallel with the specific one. With those controls, you can be sure that nothing in the extract is giving a high non-specific background and also that your primary/secondary antibodies are not producing any non-specific binding.
If the dot blot is definitely ok , then its possible that your detection antibody is biased toward recognition of the native antigen and its reactivity is absent or reduced when the antigen is denaured for western blotting procedures. This can be the case if you are using a monoclonal antibody against a conformation-sensitive epitope on the antigen. If your detection antibody is polyclonal, there are less chaces of having only conformation-sensitive antibodies in the mixture, but its still possible that the reactivity is greatly reduced on upon antigen denaturation. Are you sure your detection antibody is useful for western blot?
You have also to exclude technical reasons for western blotting failure. Do you have a positive control in this experiment (your protein of interest in purified form)? Did you check successful transfer of proteins to the membrane by protein staining? Perhaps you have to optimize the amount of total protein you are applying to WB, and the concentration of primary/secondary antibodies. The conditions that work for dot blot are not necessarily the best for WB.
My antibody is His tag antibody(HRP) and yes I have a positive control which gave a good band in western blot.
Did you use whole cell lysate in the dot blot assay and used supernatant with W.B? if yes, that suggests that your protein could be insoluble. To check that, load whole cell lysate, supernatant, and pellet for both dot blot and W.B. If you can detect your protein in the whole cell lysate and pellet but not in the supernatant in both assays that highly suggests that your protein is insoluble under your experiment conditions. If you could see your protein in any samples of dot blot but not in W.B that means your Ab can recognize the epitopes when your protein has native structure.
Best
I assume you are doing dot blot and WB with the same starting material (protein extracts), is that right?
If you are using an anti-His that works in WB in your experimental conditions, then I would suggest to check the negative controls of your dot blot to make sure there are no false positive results. If your dot blot is truly positive, perhaps your WB needs some further optimization to see a band that is not tha is not very intense. By the way, can you see a putative bad in SDS/PAGEnt?
Yes, if its a conformational protein, then it is possible that you can observe the positive result in dot blot but not in western blot.
Presence of Laemmli buffer, SDS and Beta-ME can influence denaturation, that may interrupt in antibody recognition. Try under the native gel (without SDS) and under non-reducing condition.
also, you can try optimising the salt concentration, buffer pH for better antibody recognition.
Loading concentration of your protein/antigen would be higher in case of dot blot compared to the western (and same with the primary and secondary antibodies too), that might have increase the intensity too.
Thanks,
Jwala
Gertrudis Rojas Yes, I am doing dot blot and WB with the same starting material. Actually the SDS-PAGE results for my samples are not perfect either. Here is the result of my SDS-PAGE gel.
What kind of optimization for my western blot?
You could try to load different amounts of the protein extracts in the gel, titrate the concentrations of primary and secondary antibodies and use different detection systems (ie. chemiluminiscent substrates increase sensitivity) . Its also important to check succesful transfer of the proteins to the membrane. But perhaps the WB is actually negative and the dot blot is giiving alse positive results.f
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