How do the modified amino acids work?
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.
What are the recommended final concentrations of the amino acids in the culture media?
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.
May I use only one of the photo-reactive amino acids?
For optimal crosslinking efficiency, use L-Photo-Leucine and L-Photo-Methionine in combination. If you wish to use only one of the photo-reactive 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.
When using both L-Photo-Leucine and L-Photo-Methionine, do I need to add the native analogs as well?
No, only supplement DMEM-LM medium with the photo-reactive amino acids. Adding native analogs will compete for incorporation, thereby reducing crosslinking efficiency.
Can I use a 6-Watt Lamp for photo-activation?
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.
Do photo-reactive amino acids lose mass after activation?
Yes, photoactivation of the amino acids leads to carbene free radical formation and loss of N2 gas, which is 28 Da.
Have these photo-reactive amino acids been tested in organisms other than mammalian cells?
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.
Can I use the photo-reactive amino acids to crosslink peptides?
Peptides can be synthesized using the N-termini-protected (Boc or Fmoc) photo-reactive amino acid derivatives.
My cells will not grow in DMEM, what other type of culture media can be used with the photo-reactive amino acids?
Some cells types can be adapted to grow in DMEM before using the DMEM-LM supplemented with the photo-reactive amino acids. Currently we do not offer any other leucine- and methionine-depleted culture medium.
How long do I need to incubate the photo-reactive amino acids with my cells?
Incubate the photo-reactive 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.
How stable are the photo-reactive amino acids in DMEM-LM?
Long-term stability of the photo-reactive 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 photo-reactive amino acids at -20°C as a dry compound. Just before use, add the photo-reactive amino acids to the minimal volume of DMEM-LM supplemented with dialyzed serum.
Apart from proteins, will the photo-reactive amino acids crosslink to other molecules?
Photo-reactive amino acids are incorporated into proteins during synthesis; but upon UV activation, they can crosslink to other biomolecules within proximity.