Dulbecco's Modified Eagle's Limiting Medium (DMEM-LM) - FAQs

View additional product information for Dulbecco's Modified Eagle's Limiting Medium (DMEM-LM) - FAQs (30030)

13 product FAQs found

Apart from proteins, will the Photoreactive Amino Acids crosslink to other molecules?

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

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How stable are the Photoreactive Amino Acids in DMEM-LM?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How long do I need to incubate the Photoreactive Amino Acids with my cells?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

My cells will not grow in DMEM, what other type of culture media can be used with the Photoreactive Amino Acids?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I use the Photoreactive Amino Acids to crosslink peptides?

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

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Have Photoreactive 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Do Photoreactive 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I use a 6-Watt Lamp for photoactivation of the Photoreactive Amino Acids?

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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 photoreactive amino acids. Adding native analogs will compete for incorporation, thereby reducing crosslinking efficiency.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

May I use only one of the Photoreactive 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 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

What are the recommended final concentrations of the Photoreactive 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How do the Photoreactive 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I add L-photo-leucine to Freestyle 293 Expression Medium in order to obtain a secreted protein incorporated with photo-leucine?

To use the L-photo-leucine, you will need to use a medium that is free of L-leucine. The 293 medium has all essential amino acids so they will compete with the photo amino acids for incorporation into your proteins. Our product DMEM-LM (Cat. No. 30030) is deficient in L-leucine and L-methionine.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.