Hi i'm performing dot blot immunoassay, using nylon membrane
Becaus my antibodies are anti a short peptide and not the whole native protein(and not from terminal areas) I'm afraid the native protein's conformation might cause the anti-peptide pAb to not react with it.
SO, i took 2 samples, treated one with 2% SDS and the other with 5M Urea in efforts to denature the protein. I realize now that heat denaturation might have been the better choice but it's already done and on the membrane. Also being membrane proteins i think the best way to go would be maybe to dissolve membranes in 1% triton X and then heat denaturation before blotting.
Now after i block and wash 3x with TBST do you think SDS and Urea will stay on the membrane and affect the antibody? if so, would washing more reduce the probability of that happening for example 5x?
The urea and SDS are soluble in aqueous solutions and so washed away by TBST. 3x washing is normally enough.
How did you raised your antibody? When using a peptide as an antigen, the sequence is usually selected from the segment on the surface of the parent protein. This avoids the problem you concern. You should try the native protein 1st.
Here is a good introduction on your question: http://www.ezbiolab.com/antigen_design.html
the dot blot assays did not give any positive results(native, urea, or sds) so i cannot say , but i tried tissue printing with the membrane soaked in 1% SDS after printing and the antibody successfully bound to the protein so i can confirm treating membrane with SDS will not affect reaction with normal 3x wash steps
Urea and SDS will be removed upon washing the membrane with PBST. I would suggest in future experiment for raising your PAB is to use a complex of native peptide and SDS-denatured peptide. This will lead to PABs containing external and internal epitopes. Also treat your native peptide with neutral detergent TX-100 (up to 5%) to relax secondary structure and reduce non specific binding on membrane. You can couple heat treatment with TX-100 treatment for your peptide before membrane blotting . Please refer to the following references that might explain further this issue.
Abdel-Salam, A.M. 1999. Isolation and partial characterization of a whitefly-transmitted geminivirus associated with the leaf curl and mosaic symptoms on cotton in Egypt. Arab .J. Biotech. 2(2): 193-218.
Abdel-Salam, A.M. 1999. Effect of triton X-100 inclusion in extraction buffers in reducing non-specific backgrounds in dot blot immuno-binding assay (DBIA) of plant viruses. Arab .J. Biotech. 2(1): 89-96.
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