TAMRA-Azide-PEG-biotin

Guidelines (The following is our recommended protocol. This protocol is provided for reference only and should be modified according to your specific needs).
Enrichment of N-myristoylated proteins^[1]
1. 293T cells are treated with 40 μM Alkynyl myristic acid (YnMyr) (HY-140335) for 24 h followed by lysis in PBS containing 1% Triton X-100, 0.1% SDS and protease inhibitors.
2. Proteins are diluted to 1 mg/mL with lysis buffer.
3. 100 μg of proteins are used to perform click chemistry.
4. A click mixture is prepared by adding reagents in the following order and by vortexing between the addition of each reagent:
1 μL of 10 mM capture reagent (TAMRA-Azide-PEG-biotin) (dissolved in DMSO), 2 μL of 50 mM CuSO₄, 2 μL of 50 mM TCEP (HY-W011500), 1 μL of 10 mM Tris(benzyltriazolylmethyl)amine (TBTA) (HY-116677).
5. 6 μL of the click reagent mixture is added into each protein sample followed by incubation for 1 h at room temperature with gentle votexing.
6. The click reaction is halted with 5 mM EDTA (HY-Y0682), and proteins are precipitated by adding 200 μL water, 400 μL methanol and 100 μL chloroform followed by centrifuging at 6,000 × g for 5 min.
7. After washing once with methanol, the precipitated proteins are dissolved in 10 μL PBS containing 2% SDS and 10 mM DTT (HY-15917) followed by diluted 10-fold with PBS.
8. For the affinity enrichment, the proteins are incubated with 30 μL Streptavidin Magnetic Beads (HY-K0208) for 2 h at room temperature with gentle mixing.
9. After incubation, the supernatant is collected as the unbound fraction and the beads washed 5 times with PBS containing 0.2% SDS.
10. Then the proteins are resolved by SDS-PAGE and in-gel fluorescence is scanned at 532/575 nm excitation/emission wavelengths using fully automatic luminescence imaging system followed by immunoblot.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.