Puromycin-bis(PEG2-amide)-Biotin for single-cell translationome sequencing.

Physicochemical Properties

Molecular Formula	C46H71N13O13S
Molecular Weight	1046.20
CAS #	2379889-82-2
PubChem CID	168006880
Appearance	White to off-white solid powder
Hydrogen Bond Donor Count	10
Hydrogen Bond Acceptor Count	18
Rotatable Bond Count	32
Heavy Atom Count	73
Complexity	1700
Defined Atom Stereocenter Count	8
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Puromycin-bis(PEG2-amide)-Biotin shares the same target as puromycin, which is the ribosomal A site. It binds to the ribosome and mimics the 3' end of aminoacyl-tRNA, leading to premature termination of protein synthesis.
ln Vitro	Puromycin-bis(PEG2-amide)-Biotin efficiently incorporated into nascent polypeptides in cultured cells, as detected by streptavidin-based pull-down assays and mass spectrometry. This demonstrated its ability to label newly synthesized proteins for subsequent purification and analysis [1]
ln Vivo	In C57BL/6 mice, intraperitoneal injection of Puromycin-bis(PEG2-amide)-Biotin (50 mg/kg) resulted in labeling of newly synthesized proteins in multiple tissues. Tissues with high protein synthesis rates, such as the intestinal epithelium and bone marrow, showed strong biotin signals detected by streptavidin-fluorophore conjugation. Moderate signals were observed in the liver and kidney, reflecting baseline protein synthesis levels [1] The biotinylated protein signal peaked at 1 hour post-injection and remained detectable for up to 4 hours, enabling temporal analysis of in vivo protein synthesis dynamics [1]
Enzyme Assay	Ribosome Binding Assay: Radiolabeled Puromycin-bis(PEG2-amide)-Biotin was incubated with purified ribosomes in buffer containing Mg²⁺ and ATP. Binding was measured by nitrocellulose filter retention, showing specific interaction with ribosomes. Competition assays with unlabeled puromycin confirmed the binding specificity [1]
Cell Assay	rotein Labeling and Purification: Cells were treated with Puromycin-bis(PEG2-amide)-Biotin (1–10 μM) for 30–60 minutes, followed by lysis and streptavidin bead purification. Western blot analysis confirmed the enrichment of biotinylated proteins, indicating successful labeling of newly synthesized polypeptides [1]
Animal Protocol	- In Vivo Protein Labeling in Mice: Puromycin-bis(PEG2-amide)-Biotin (50 mg/kg) was administered intraperitoneally to C57BL/6 mice. After 1 hour, tissues were harvested and processed for streptavidin-based detection. Fluorescence imaging showed labeling in highly proliferative tissues like the intestine and bone marrow [1] - Drug Formulation: Puromycin-bis(PEG2-amide)-Biotin was dissolved in sterile DMSO and diluted with PBS to a final concentration of 10 mg/mL before injection [1]
References	[1]. Yungui Yang, et al. Single-cell translatome sequencing method. WO2023284076A1.
Additional Infomation	- Puromycin-bis(PEG2-amide)-Biotin is a modified puromycin analog with two PEG2-amide linkers and a biotin moiety, enabling bioorthogonal labeling of newly synthesized proteins for downstream applications such as proteomics and single-cell translatome sequencing [1] - The compound combines the protein synthesis inhibitory activity of puromycin with the high-affinity binding of biotin to streptavidin, allowing efficient capture and analysis of nascent polypeptides in complex biological samples [1]

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	0.9558 mL	4.7792 mL	9.5584 mL
	5 mM	0.1912 mL	0.9558 mL	1.9117 mL
	10 mM	0.0956 mL	0.4779 mL	0.9558 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.