On 2017 Sep 08, Youhe Gao commented:

A strategy named 4F-acts was proposed a few years ago trying to minimize false positives and false negatives. Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde crosslinking can fix transient and weak protein interactions. With brief exposure to a high concentration of formaldehyde during the crosslinking, the complex is crosslinked only partially, so that the complex is small enough to be resolved by SDS-PAGE, and the uncrosslinked parts of the proteins can be used for identification by shotgun proteomics. Immunoaffinity purification can Fish out complexes that include the proteins of interest. Because the complex is covalently bound, it can be washed as harshly as the antibody-antigen reaction can stand; the weak interactions will remain. Even if the nonspecific binding can persist on the beads or antibody, it will be eliminated at the next step. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncrosslinked complexes and simultaneously providing molecular weight information for identification of the complex. The SDS-polyacrylamide gel can then be sliced on the basis of the molecular weight without staining. All the protein complexes can be identified with the sensitivity of mass spectrometry rather than sensitivity of the staining method. The advantages are the following: (i) The method does not involve tagging. (ii) It does not include overexpression. (iii) A weak interaction can be detected because the complexes can be washed as hard as the antigen-antibody reaction can stand as the complexes are crosslinked covalently. No new covalent bond can form as a false positive result. (iv) The formaldehyde crosslinking can be performed at the cellular, tissue, or organ level fast enough so that the protein complexes are fixed in situ in real time. Proteome Science 2014, 12:6 doi:10.1186/1477-5956-12-6

On 2017 Sep 08, Youhe Gao commented:

A strategy named 4F-acts was proposed a few years ago trying to minimize false positives and false negatives. Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde crosslinking can fix transient and weak protein interactions. With brief exposure to a high concentration of formaldehyde during the crosslinking, the complex is crosslinked only partially, so that the complex is small enough to be resolved by SDS-PAGE, and the uncrosslinked parts of the proteins can be used for identification by shotgun proteomics. Immunoaffinity purification can Fish out complexes that include the proteins of interest. Because the complex is covalently bound, it can be washed as harshly as the antibody-antigen reaction can stand; the weak interactions will remain. Even if the nonspecific binding can persist on the beads or antibody, it will be eliminated at the next step. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncrosslinked complexes and simultaneously providing molecular weight information for identification of the complex. The SDS-polyacrylamide gel can then be sliced on the basis of the molecular weight without staining. All the protein complexes can be identified with the sensitivity of mass spectrometry rather than sensitivity of the staining method. The advantages are the following: (i) The method does not involve tagging. (ii) It does not include overexpression. (iii) A weak interaction can be detected because the complexes can be washed as hard as the antigen-antibody reaction can stand as the complexes are crosslinked covalently. No new covalent bond can form as a false positive result. (iv) The formaldehyde crosslinking can be performed at the cellular, tissue, or organ level fast enough so that the protein complexes are fixed in situ in real time. Proteome Science 2014, 12:6 doi:10.1186/1477-5956-12-6