L-Photo-Methionine - FAQs

Photoreactive amino acids are incorporated into proteins during synthesis; but upon UV activation, they can crosslink to other biomolecules within proximity.

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Long-term stability of the photoreactive amino acids in media has not been determined but should be comparable to their natural analogs, if protected from light. For best results, store the photoreactive amino acids at -20°C as a dry compound. Just before use, add the photoreactive amino acids to the minimal volume of DMEM-LM supplemented with dialyzed serum.

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Incubate the photoreactive amino acids in DMEM-LM with cells for 24 hours. For proteins with high turnover, a minimum incubation of 8 hours is needed for detectable crosslinking levels. Incubation for longer than 24 hours might significantly affect cell growth or viability.

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Some cells types can be adapted to grow in DMEM before using the DMEM-LM supplemented with the photoreactive amino acids. Currently we do not offer any other leucine- and methionine-depleted culture medium.

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Peptides can be synthesized using the N-termini-protected (Boc or Fmoc) photoreactive amino acid derivatives.

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Both leucine and methionine are essential mammalian amino acids that can be substituted by these photo-reactive analogs. We have not tested cells from other organisms.

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Yes, photoactivation of the amino acids leads to carbene free radical formation and loss of N2 gas, which is 28 Da.

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For best results, use UV lamps that irradiate from 320 to 370 nm with an 8-watt minimum output. Using lower-wattage hand-held lamps will result in lower crosslinking efficiencies.

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No, only supplement DMEM-LM medium with the photoreactive amino acids. Adding native analogs will compete for incorporation, thereby reducing crosslinking efficiency.

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For optimal crosslinking efficiency, use L-Photo-Leucine and L-Photo-Methionine in combination. If you wish to use only one of the photoreactive amino acids, supplement DMEM-LM media with 105 mg/L of tissue-culture grade L-Leucine or 30 mg/L of L-methionine, depending on which amino acid is deficient.

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For best results, use 2 mM L-Photo-Methionine and 4 mM L-Photo-Leucine. These concentrations may be reduced when using cell types that do not tolerate such levels.

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L-Photo-Leucine and L-Photo-Methionine are amino acid analogs of L-leucine and L-methionine that are endogenously incorporated into the primary sequence of proteins during synthesis and then activated by ultraviolet (UV) light to covalently crosslink within protein-protein interaction domains. The powerful method enables characterization of stable and transient protein interactions without using completely foreign chemical crosslinkers and associated solvents that can adversely affect the native environment.

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L-photo-leucine (Cat no. 22610) and L-photo-methionine (Cat no. 22615) are amino acid derivatives that possess diazirine rings for UV photo-crosslinking of proteins. When used in combination with specially formulated limiting media, these photo-activatable derivatives of leucine and methionine are treated like the naturally occurring amino acids by the protein synthesis machinery within the cell. As a result, they can be substituted for leucine or methionine in the primary sequence of proteins during synthesis. 

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