The photoreactive amino acids are metabolically incorporated into the expressed proteins of actively growing cells by incubating cells in limiting media without methionine and leucine but containing Photo-Leucine and Photo-Methioinine amino acids.
Proteins containing the photoreactive analogs readily crosslink upon activation with UV light (365 nm) using a common laboratory UV lamp (Figure 2 and Table).
Crosslinked protein complexes can be detected by reduced SDS-PAGE mobility followed by Western blotting.
In Vivo Crosslinking of Live Cells
To demonstrate photoreactive crosslinking in live cells, HeLa cells were treated with Photo-Leucine and Photo-Methionine before photoactivation with UV light. For comparison, HeLa cells were also crosslinked using formaldehyde, DSG, and DSS. Treated cells were then lysed and analyzed by SDS-PAGE and Western blotting using an antibody against the homodimeric transcription factor Stat3.
Samples were normalized for protein amount and probed with antibodies against GAPDH and beta-actin to verify equal loading. Addition of the photoreactive amino acids did not adversely affect overall protein levels or antibody epitope recognition after Western blotting (Figure 3A).
Formaldehyde was unsuccessful in crosslinking any Stat3 dimers and even resulted in reduced Stat3 monomers, most likely caused by decreased solubility of Stat3 after nonspecific crosslinking. For the photoreactive amino acids, crosslinked products were easily observed in UV-treated samples compared to mock-treated controls. The homobifunctional NHS esters DSG and DSS were also able to efficiently crosslink Stat3. They, however, produced dimers having slightly different SDS-PAGE mobility than the photoreactive amino acid-crosslinked product.
This difference may be caused by the various amino acids involved in crosslinking: leucine and methioinine vs. lysine and arginine. Consequently, photoreactive amino acids and NHS esters are complementary crosslinking approaches; one or the other may work better depending on the amino acids required for specific protein interaction domains.
To quantify and compare the effects of the different crosslinking methods on protein solubility, the protein concentrations of the resulting cell lysates were determined using the Thermo Scientific Pierce® BCA protein assay and normalized to mock-treated controls.