Thermo Scientific™ Dulbecco's Modified Eagle's Limiting Medium (DMEM-LM)

Description

• Thermo Scientific™ Pierce Photoreactive Amino Acids are analogs of L-Leucine and L-Methionine that can be incorporated into proteins during synthesis and then light-activated to covalently crosslink interacting proteins in cells
• Thermo Scientific L-Photo-Leucine and L-Photo-Methionine are analogs of L-Leucine and L-Methionine amino acids that have activatable diazirine side chains capable of chemical crosslinking to adjacent molecules when exposed to ultraviolet light
• Like their naturally occurring counterparts, these photo-reactive amino acids can be endogenously incorporated into the primary sequence of proteins during synthesis
• Protein-protein interaction domains involving the synthesized proteins in their native environments can then be covalently crosslinked by activating the diazirine groups
• Traditional crosslinking experiments require adding bifunctional chemical crosslinkers and associated reagent solvents, which can adversely affect the cell biology being studied
• Using photo-reactive amino acids minimizes these potentially adverse effects and enables both stable and transient protein interactions in cells to be studied and characterized with a high degree of confidence.When used in combination with specially formulated limiting cell media that is devoid of leucine and methionine, the photo-activatable derivatives are treated like naturally occurring amino acids by the protein synthesis machinery
• As a result, they can be substituted for leucine or methionine in the primary sequence of proteins
• When exposed to UV light the diazirine rings become reactive intermediates that form covalent bonds with nearby protein side chains and backbones
• Naturally associating binding partners are then instantly trapped
• Crosslinked protein complexes are detected by decreased mobility on SDS-PAGE followed by Western blot detection, size-exclusion chromatography, sucrose density-gradient sedimentation or mass spectrometry
• Highlights: In vivo labeling – incorporate photo-reactive group into proteins using normal cellular machinery In vivo crosslinking – find interacting proteins in the native cellular environment Increased specificity compared to traditional methods – crosslink interacting proteins correctly positioned at their interfaces within protein interaction domains Complementary to traditional methods – provides detailed characterization of protein interactions when compared to results with formaldehyde and other more general crosslinking approaches Efficient recovery – 90% protein recovery in cell lysates after crosslinking Compatible – crosslink proteins expressed in a wide variety of cell lines, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3 and C6 Easy to use – reagents are photo-stable under normal laboratory lighting, eliminating the need to work in the dark