Small molecules for monitoring protein synthesis

Small molecules

The characterization of unexplored translational control mechanisms are now feasible with our selection of small molecules to study protein synthesis and ribosome-associated binding proteins.


OPP (O-propargyl-puromycin) is an alkyne analog of puromycin that is efficiently incorporated into newly translated proteins in complete methionine-containing media

aaaaaaaaaaaaaaaaa

For research use only!
Shipping: shipped on blue ice
Quantity: 3 mg
Storage Conditions: Store at -20 °C. One week to ambient temperature possible.
Shelf Life: 6 months after the date of delivery
Molecular Weight: 495.53 g/mol (free acid)
CAS#: 1416561-90-4
Purity: ≥ 95 % (HPLC)
Form: colorless to slightly white solid
Solubility: DMSO, PBS (up to 50 mM tested)
Spectroscopic Properties: λmax 275 nm; ε 20.0 L mmol-1 cm-1

Applications:
Protein synthesis monitoring in cell culture and whole organisms[1,2]

Description:

Liu et al.[1] reported a non-radioactive alternative to analyze newly synthesized proteins in cell culture and whole organisms that is based on O-Propargyl-puromycin, an alkyne analog of puromycin.
O-Propargyl-puromycin is cell-permeable and incorporates into the C-terminus of translating polypeptide chains thereby stopping translation.
The resulting truncated C-terminal alkyne labeled proteins can subsequently be detected via Cu(I)-catalyzed click chemistry that offers the choice to introduce a Biotin group (via Azides of Biotin) for subsequent purification tasks or a fluorescent group (via Azides of fluorescent dyes) for subsequent microscopic imaging.
In contrast to Azidohomoalanine (AHA) or Homopropargylgycine (HPG) based non-radioactive methionine analog-approaches, methionine free-medium is not required for O-Propargyl-purmoycin-based monitoring of nascent protein synthesis.
Presolski et al.[4] and Hong et al.[5] provide a general protocol for Cu(I)-catalyzed click chemistry reactions that may be used as a starting point for the set up and optimization of individual assays.

Selected References:
[1] Liu et al. (2012) Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl. Acad. Sci. USA 109 (2):413.
[2] Signer et al. (2014) Haematopoietic stem cells require a highly regulated protein synthesis rate. Nature 509:49.
[3] Grammel et al. (2013) Chemical reporters for biological discovery. Nat. Chem. Biol. 9 (8):475.
[4] Presolski et al. (2011) Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation. Current Protocols in Chemical Biology 3:153.
[5] Hong et al. (2011) Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angew. Chem. Int. Ed. 48:9879.


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

aaaaaaaaaaaa

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. (2019) Sensing translation activity at the ribosome surface with UV-active small molecules. ACS-Omega


3PC 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).

aaaaaaaaaaa

For research use only!
Shipping: shipped on dry ice
Quantity: 10 µL DMSO – 100 mM
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.

Selected References:
[1] Kandala D.T. et al. (2019) Sensing translation activity at the ribosome interface with UV-active small molecules. ACS-Omega


Click kit for azide-alkyne cycloaddition

For research use only!

Shipping: shipped at ambient temperature
Storage Conditions: store components as indicated on the data sheet
Shelf Life: 6 months after the date of delivery

Description:

The Click Chemistry Capture Kit provides all necessary reagents to covalently link specific alkyne probes by a Cu(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) with the azide-PEG-biotin followed by pull-down of the tagged proteins by magnetic beads.  After separation, proteins can be processed for mass spectroscopy (e.g. LC-MS/MS) or immunoblotting analysis.

Content (6 reactions):

  • Lysis buffer – store at 4 °C
  • Additive 1- store at 4 °C
  • Copper (II) Sulfate (100 mM) – store at ambient temperature
  • Additive 2 – store at ambient temperature
  • azide-PEG-biotin
  • Buffer W
  • eMagSi magnetic beads

Step 1: Preparation of the lysis buffer

Keep the required optimal volume of lysis buffer on ice and add the following components (not provided): sodium deoxycholate (1% final concentration), 5 U/mL DNase I and 200 U/mL RiboLock RNase Inhibitor.

Step 3a: Reaction of the alkyne UV-active probe in complete media

  • Treat the cells with the alkyne UV-active probe for 10 min at  37°C.
  • Wash with cold PBS
  • Placed on ice and irradiated under a UV lamp (BLX-365, 5 x 8 W) at 365 nm for 5 min (0.75 J/cm2), followed by lysis.

Step 3b: Reaction in the cell lysate

  • Lysis and precipitation of the nuclei and cellular debris by centrifugation at 18000 g and 4°C for 5 min
  • Collect the supernatant and dilute it to A260 = 1 a.u/µL with buffer W (10 mM NaCl, 10 mM MgCl2, 20 µg/mL cycloheximide, and 10 mM Hepes, pH 7 in DEPC water) to a final volume of 150 µL.
  • Incubated with the reactive probe for 1 hour in a 12-well plate
  • UV-irradiate at 365 nm for 5 min (0.75 J/cm2).

Step 4: Washing

After incubation, place the cells on ice and wash them with cold PBS containing CHX (20 mg/mL)
Remove residual PBS with a pipet

Step 5: Lysis

 

Step 6: Copper-catalyzed “click” reaction in the cell lysates and pull-down assays

Incubate the cell lysate (0.35 mL)  with CuSO4 (2.00 µmol), azide-PEG-biotin (1:1 mole with the alkyne probe), additive 1 (10 µmol) and additive 2 (100 µmol) overnight a 4°C. For the pull-down, cell lysates were then incubated with 50-100 µL of eMagSi

Small molecules
IMMAGINA BioTechnology’s General Terms and Conditions