6 replies to this topic

[Shelley7]

Does anyone know if it is possible to precipitate proteins after immunohistochemistry. We have an antibody that works well in situ but may not work to precipitate protein from lysates. Is it possible to permeabilze cells, expose to antibody under natural conditions, and then precipitate out the antibody complexes? Standard Co-IP precipitates proteins but not the one we are interested in, however, the antibody works great to identify our protein in fixed cells in culture.

THANKS!!

[bob1]

No, you can not precipitate fixed proteins, they are crosslinked to other proteins and cellular components by the fixation step. This is why the proteins stay in place during ICC/IHC rather than diffusing/washing out of the cell.

It is possible to IP native proteins though, use a lysis buffer that contains little or no detergent and is very mildly hypotonic, and disrupt the cells with a glass homogeniser such as the one in the attached file.

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[Helios]

i agree with bob....you can't do IP's for fixed tissues as the proteins are already cross linked.....what you can do is try to try your antibody concentration required to immunoprecipitate protein from the lysate because the these conc are different for IHC and IP.

[Shelley7]

bob1, on Nov 10 2009, 03:21 PM, said:

No, you can not precipitate fixed proteins, they are crosslinked to other proteins and cellular components by the fixation step. This is why the proteins stay in place during ICC/IHC rather than diffusing/washing out of the cell.

It is possible to IP native proteins though, use a lysis buffer that contains little or no detergent and is very mildly hypotonic, and disrupt the cells with a glass homogeniser such as the one in the attached file.

[Starlight83]

bob1, on Nov 11 2009, 12:21 AM, said:

No, you can not precipitate fixed proteins, they are crosslinked to other proteins and cellular components by the fixation step. This is why the proteins stay in place during ICC/IHC rather than diffusing/washing out of the cell.

It is possible to IP native proteins though, use a lysis buffer that contains little or no detergent and is very mildly hypotonic, and disrupt the cells with a glass homogeniser such as the one in the attached file.

Please, can you give us the recipe of this lysis buffer?
I want to lyse neurons perturbing as little as possible protein-protein interaction

[bob1]

Try buffer A in attached paper.

Attached File(s)

•  NAR_cellular_fractionation_paper.pdf (259.45K)
  Number of downloads: 10

[Starlight83]

bob1, on Nov 13 2009, 12:27 AM, said:

Try buffer A in attached paper.

Thank you very much !!!!