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| Targets |
Rhodopsin Epitope Tag is a peptide tag that does not have a biological target in the context of a drug. Instead, it is a molecular tool that is recognized by specific anti-rhodopsin antibodies (e.g., monoclonal antibody Rho-1D4). The tag is genetically fused to the N-terminus, C-terminus, or internal region of a protein of interest via recombinant DNA technology. The epitope tag serves as an antigenic determinant that allows the fusion protein to be detected, purified, or immunoprecipitated using anti-rhodopsin antibodies. This system does not target any cellular pathway; rather, it is a tool for studying protein expression, localization, interaction, and function. The tag is small (9 amino acids) and is generally considered to have minimal impact on the structure or function of the fusion protein.
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| ln Vitro |
A 9-amino acid peptide called Rhodopsin Epitope Tag is found in the C-terminal region of bovine rhodopsin. Many anti-rhodopsin antibodies can identify the Rhodopsin Epitope Tag, which is widely used as an epitope tag. Synthetic peptides have been used to define the specificities of four monoclonal antibodies, rho 1D4, 1C5, 3A6, and 3D6, that were produced by immunization of rod outer segments containing rhodopsin. All of these antibodies recognize linear antigenic determinants of 4–11 residues and interact within the 18 residues at the COOH terminus of rhodopsin [1].
Rhodopsin Epitope Tag does not possess intrinsic biological activity; it is a chemical tool used in in vitro biochemical and cell biology assays. In vitro, the peptide can be used as a positive control or blocking peptide in immunodetection assays. For example, in an ELISA or Western blot, the synthetic peptide (Rhodopsin Epitope Tag) can be used to compete with a tagged fusion protein for antibody binding, thereby confirming the specificity of the antibody-antigen interaction. When the peptide is pre-incubated with the anti-rhodopsin antibody, it blocks binding to the epitope-tagged protein, reducing the signal. This serves as a specificity control. Additionally, the peptide can be conjugated to carrier proteins (e.g., KLH, BSA) for the generation of polyclonal antibodies against the tag. In the context of cellular assays, the tag does not affect cell viability or signaling. It is considered inert and is used exclusively as a detection handle. |
| Enzyme Assay |
A typical non-cellular (cell-free) protocol for using Rhodopsin Epitope Tag as a control in an ELISA is as follows. A 96-well plate is coated with 100 uL per well of a solution containing 1 ug/mL of the epitope-tagged fusion protein in PBS (pH 7.4) and incubated overnight at 4degC. The plate is washed three times with PBS-T (PBS containing 0.05% Tween-20) and blocked with 3% BSA in PBS-T for 1 hour at room temperature. For competition assays, the primary antibody (anti-rhodopsin, e.g., Rho-1D4, 1:1000 dilution) is pre-incubated with increasing concentrations of the synthetic Rhodopsin Epitope Tag peptide (0.1-100 ug/mL) for 1 hour at room temperature. The antibody-peptide mixture is then added to the wells (100 uL/well) and incubated for 1-2 hours at room temperature. The plate is washed three times with PBS-T. A secondary antibody (HRP-conjugated anti-mouse or anti-rabbit, as appropriate) diluted 1:5000 in 1% BSA-PBS-T is added (100 uL/well) and incubated for 1 hour. After washing, 100 uL of TMB substrate is added, and the plate is incubated for 5-15 minutes in the dark. The reaction is stopped with 50 uL of 1 M H2SO4, and the absorbance is read at 450 nm. The percentage of competition is calculated relative to the control without peptide. The peptide should block antibody binding in a concentration-dependent manner, confirming the specificity of the antibody for the epitope.
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| Cell Assay |
A typical in vitro cellular protocol for using Rhodopsin Epitope Tag involves its use as a tag for fusion proteins expressed in mammalian cells. A target gene of interest is cloned into a mammalian expression vector containing the sequence for the Rhodopsin Epitope Tag (e.g., pCDNA3.1-RhoTag). The construct is verified by sequencing. Human embryonic kidney (HEK293T) cells are seeded in 6-well plates at 5×10⁵ cells/well in DMEM with 10% FBS at 37degC in 5% CO2 for 24 hours. The cells are transfected with 2 ug of the plasmid DNA using a transfection reagent (e.g., Lipofectamine 3000) according to the manufacturer‘s protocol. After 48-72 hours, the cells are harvested, washed with PBS, and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors) on ice for 30 minutes. The lysate is clarified by centrifugation at 14,000 g for 15 minutes at 4degC. The supernatant is used for Western blot analysis: samples are separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% non-fat milk, and probed with anti-rhodopsin antibody (Rho-1D4, 1:1000-1:5000) overnight at 4degC, followed by an HRP-conjugated secondary antibody. Chemiluminescent detection is performed. For immunoprecipitation, the cleared lysate is incubated with anti-rhodopsin antibody (2-5 ug) and protein A/G beads for 2-4 hours at 4degC, then washed, eluted with 2× SDS loading buffer, and analyzed by Western blot. The tag also allows for immunofluorescence localization of the fusion protein in cells fixed with 4% paraformaldehyde.
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| Animal Protocol |
An in vivo animal protocol for Rhodopsin Epitope Tag is not standard, as the tag is used for protein detection in vitro or ex vivo, not for administration as a therapeutic. However, if one wishes to evaluate the expression of a Rhodopsin-tagged therapeutic protein in an animal model, a typical protocol involves intravenous or intraperitoneal administration of the tagged protein (e.g., a tagged antibody or enzyme) to mice. For example, female BALB/c mice (6-8 weeks old) are injected intravenously with 1-10 mg/kg of a recombinant fusion protein containing the Rhodopsin Epitope Tag. At various time points after injection (e.g., 0.5, 1, 2, 4, 8, 24, 48 hours), blood samples are collected from the tail vein into EDTA tubes. Plasma is separated by centrifugation and stored at -80degC. Animals are euthanized at the final time point, and tissues (e.g., liver, kidney, spleen, lung) are harvested, snap-frozen, and homogenized in RIPA buffer. The plasma samples and tissue lysates are analyzed by Western blot or ELISA using the anti-rhodopsin antibody to detect the tagged protein. The amount of protein is quantified using a standard curve of the same purified protein. The elimination half-life, area under the curve (AUC), and other pharmacokinetic parameters are calculated. This approach allows for tracking of the protein‘s distribution and clearance.
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| ADME/Pharmacokinetics |
Pharmacokinetic (PK) data for the Rhodopsin Epitope Tag itself are not applicable, as the tag is a peptide that is not administered alone. When the tag is genetically fused to a protein, the PK properties of the fusion protein are determined by the carrier protein, not by the epitope tag. The 9-amino acid tag is small and is unlikely to alter the pharmacokinetics of the parent protein significantly. The tag may be immunogenic in vivo if the animal has been previously exposed to bovine rhodopsin, but in most experimental settings, the tag is considered inert. For research purposes, the tag is used as a detection tool, not as a pharmacokinetic modifier.
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| Toxicity/Toxicokinetics |
The Rhodopsin Epitope Tag peptide itself (9-mer) is generally considered non-toxic. It is a synthetic peptide and, at the concentrations used in vitro (ug/mL to mg/mL), does not cause cytotoxicity. In vivo, when administered as part of a fusion protein, the tag does not contribute to toxicity. Standard laboratory safety precautions should be followed when handling the synthetic peptide, including the use of gloves, lab coats, and safety glasses. The lyophilized peptide should be stored at -20degC, protected from light and moisture. Once reconstituted in water or buffer, the peptide should be stored in aliquots at -20degC to avoid repeated freeze-thaw cycles. It is for research use only and is not intended for human therapeutic, diagnostic, or clinical applications.
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| References |
[1]. Hodges RS, Heaton RJ, Parker JM, Molday L, Molday RS. Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin. J Biol Chem. 1988;263:11768–11775.
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| Additional Infomation |
Rhodopsin Epitope Tag is a 9-amino acid peptide (TETSQVAPA) derived from the C-terminal region of bovine rhodopsin. It is recognized by anti-rhodopsin antibodies, most notably the monoclonal antibody Rho-1D4. The tag is widely used in molecular biology for the detection, purification, and immunoprecipitation of recombinant fusion proteins. Its small size (9 aa, MW ~903 Da) minimizes interference with protein function. The tag can be fused to the N- or C-terminus of a target protein. It is also used as a positive control or blocking peptide in immunodetection assays. The molecular formula is C37H62N10O16, and the molecular weight is 902.95. It is supplied as a lyophilized solid with ≥95% purity. The product is for research use only and is not approved for clinical or diagnostic applications.
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| Exact Mass |
902.435
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| CAS # |
102099-07-0
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| PubChem CID |
134611628
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| Appearance |
Typically exists as solid at room temperature
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| Hydrogen Bond Donor Count |
14
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| Hydrogen Bond Acceptor Count |
17
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| Rotatable Bond Count |
26
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| Heavy Atom Count |
63
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| Complexity |
1700
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| Defined Atom Stereocenter Count |
11
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| SMILES |
C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)O)N)O
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| InChi Key |
YNNNDDQTLPJPIA-KCMKZVLYSA-N
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| InChi Code |
InChI=1S/C37H62N10O16/c1-15(2)27(34(59)40-16(3)36(61)47-13-7-8-23(47)32(57)41-17(4)37(62)63)45-29(54)20(9-11-24(38)51)42-31(56)22(14-48)44-35(60)28(19(6)50)46-30(55)21(10-12-25(52)53)43-33(58)26(39)18(5)49/h15-23,26-28,48-50H,7-14,39H2,1-6H3,(H2,38,51)(H,40,59)(H,41,57)(H,42,56)(H,43,58)(H,44,60)(H,45,54)(H,46,55)(H,52,53)(H,62,63)/t16-,17-,18+,19+,20-,21-,22-,23-,26-,27-,28-/m0/s1
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| Chemical Name |
(4S)-5-[[(2S,3R)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[(2S)-2-[[(1S)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-4-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]-5-oxopentanoic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O: 12.5 mg/mL (13.84 mM)
DMSO: 2.86 mg/mL (3.17 mM) |
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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