RiboNext 3PB (photo-active/ jeff-alkyne puromycin)

3PB is a UV-active alkyne analog of puromycin that efficiently binds to a network of proteins involved in ribosome function and activity (e.g. eEF1A, ENO1, GRP78).

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Procedure:

  1. Add the drug to growing cells in complete media and incubate for 10 min before photoactivation and cell lysis.
  2. Remove media containing photo-amino acids from cells and wash twice with PBS.
  3. Add sufficient PBS to completely cover cells to prevent drying during UV irradiation.
  4. Position cells 1-5 cm from UV bulbs and irradiate for five minutes or for a total of 0.75 J/cm2.
  5. Rotate dish during irradiation for even activation.
  6. Harvest cells for lysis and analyze crosslinked proteins by Western blot or other method

For research use only!
Shipping: shipped on dry ice
Quantity: 3 mg
Storage Conditions: Store at -80 °C.
Shelf Life: 3 months after the date of deliveryMolecular
Purity: ≥ 95 % (HPLC)
Form: DMSO solution
Solubility: DMSO, PBS (up to 100 µM tested)
Spectroscopic Properties: λmax 275 nm

Applications:
Monitoring of Protein synthesis activity – pull down of ribosome-associated proteins

Description:
A useful tool for sensing translation efficiency and selective deep-sequencing RNA analysis.

Photoactivation Information:

Use a UV lamp that irradiates from 320 to 370 nm for photoactivation. High wattage lamps are more effective and require shorter exposure times than low wattage lamps. Suggestions for lamps include the Stratalinker 2400 (5 × 15 watt bulbs, emission at 365 nm), mercury vapor lamps (200 watt, 300-360 nm), and Spectroline or UVP handheld lamps (8 watt, emission at 365 nm). Using lower-wattage hand-held lamps, such as 6 watt lamps, will result in lower crosslinking efficiencies. Note: The optimal wavelength for photoactivation is 345 nm. Do not use UV lamps that emit light at 254 nm as this wavelength causes proteins and DNA and RNA to photodestruct. Filters that remove light at wavelengths below 300 nm are necessary for mercury vapor lamps. • Perform UV irradiation in a shallow, uncovered reaction vessel/plate for maximum efficiency. Irradiation efficiency decreases logarithmically with increased distance from the light source. Position a UV lamp 3-5 cm from cells for 15 watt lamps. For lower-powered, hand-held lamps, use a distance of 1 cm without filter, if possible. For lamps > 150 watts, use a distance of 20 cm with a 300 nm filter. Perform photoactivation by placing the lamp above the open reaction vessel to avoid impeding irradiation by the vessel. Samples may need to be rotated for even UV irradiation. Use a total UV irradiation time less than 7 minutes for in vivo crosslinking of live cells.

Selected References:
[1] Kandala D.T. et al. (2018) Sensing translation activity at the ribosome surface with UV-active small molecules. BioRxiv