Antibodies and triton X - (Jul/31/2007 )
Maybe somebody can help me
I have read several immunofluorescence protocols where the incubation of primary antibodies is done in the presence of Triton X. Is this necessary?
Have you ever heard that an antibody needs to be incubated in any other detergent in order to work? Why is that?
thank you all
we have a protocol for immunofluorescence where we add triton to all solutions but its conc. is very low (0.01%). I guess this is to decrease the surface tension between the coverslips and cells so that cells donot come off the coverslip. Its not necessary to have triton but if the cells are not well attached, this might help.
Thank you for your answer scolix.
If you had different results using Methanol or Triton in you immunofluorescence assays, what technique would you believe in?
I have tried both with and without triton for immunofluorescence. Both work the same. I havn't used methanol, so I cannot judge it.
Methanol is for fixation, although it might cause holes in cell membrane --> somewhat permeabilize cells, but after methanol fixing, we still add in a permeabilization step, using either TritonX-100 or saponin. As said, Triton is for permeabilization. So you should not compare methanol method (fixation) with Triton (only permeabilization). Also, the fixation method of course could affect the results. Some antibodies may work well with methanol, some may not. Furthermore, the fixing method can also affect the cell structure. For example, if you are detecting Golgi proteins such as GM130, methanol fixation is fine; but if you are checking endosomal proteins such as EEA1, methanol is out of question.