I have recently performed a Western blot with the aim of detecting some allergens. Clear bands were resulted in SDS-PAGE and complete transfer of the bands to the membrane (NCP) was observed. I used allergic patients IgE as primary antibody and incubated the membrane overnight at 4C. My secondery antibody was anti human IgE conjugated with HRP and the condition of secondary incubation was 2 h at 4C. In detection step 4-Chloro-1-naphthol was used as the substrate which was activated with H2O2. However, no bands were resulted after detection step. Is there any particular reason for not detecting the bands in the membrane?
There are several things to note in your experiment.
1. The antibodies produced against the allergen in the patients might be to the 3-D epitopes of the allergen. So your SDS-PAGE denatures your allergen and you can not detect the bands. You need to run a native gel or for better detection, you need to develop ELISA for the same.
2. You need to have a positive control. Are there any commercially available primary antibodies for the allergen?
3. You might also encounter basic western blot problems, which you can test using other antigen and antibody setup.
I hope this information is useful to you. Goodluck
There are several things to note in your experiment.
1. The antibodies produced against the allergen in the patients might be to the 3-D epitopes of the allergen. So your SDS-PAGE denatures your allergen and you can not detect the bands. You need to run a native gel or for better detection, you need to develop ELISA for the same.
2. You need to have a positive control. Are there any commercially available primary antibodies for the allergen?
3. You might also encounter basic western blot problems, which you can test using other antigen and antibody setup.
I hope this information is useful to you. Goodluck
Dear Budida,
Thank you for the suggestions,I did check for basic errors with another antigen antibody set up as you mentioned in point no. 3 and bands were detected. And there are no commercially available primary anti bodies for my allergens.
The primary antibody concentration in the patient serum might be low. Try to concentrate the patient sera and try in different dilutions.
The incubation time and temperature of secondery antibody can be 2h at 37C, which works in my WB. You can have a try. BTW, it's better to have a positive control.
Hello,
Sorry, I can't help you because I have the same problem. But in my case, I am using some centromeric proteins as the antigens. I have obtained clear bands of my antigens in SDS-PAGE and the transfer of the bands to the nitrocellulose membrane was observed. My primary antibodies (IgG) was sera of patients with autoimmune disease, and my secondary antibody was anti-human IgG conjugated with HRP. I tried the detection step using the TMB substrate once time and Chloronaphtol another time. Unfortunately, no bands!!!
I don't know what is the problem exactly!
Additional information
As Giriprakash suggested, the specific antibodies in the serum might be too low to be used for your detection system in addition to their inability to bind to a denatured protein in SDS PAGE. The presence of antibodies in the host may be low if the patient is not exposed to the allergen or the antigen recently. In this case detection with SDS PAGE may not work. I assume it is not a single problem but a combination of problems where solving one problem may not give you a positive result.
I suggest you go for a native gel electrophoresis. Or ELISA is a lot sensitive and quicker detecting antibodies in the serum.
I hope this information is useful to you. Goodluck
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