Vari Fluor 405 SE (VF 405 SE)

Preparation of stock solutions 1. Preparing proteins Please get ready the protein (antibody) concentration to 2 mg/mL for the optimal labeling effect. 1) The protein solution's pH should be 8.5±0.5. Using 1 M sodium bicarbonate, correct the pH if it is less than 8.0. 2) The labeling efficiency will be significantly decreased if the protein content is less than 2 mg/mL. It is advised that the final protein concentration range be between 2 and 10 mg/mL in order to achieve optimal labeling efficiency. 3) To ensure optimal labeling efficacy, the protein needs to be in a buffer free of ammonium ions and primary amines, like Tris or glycine. 2. Dye preparation To create a stock solution of 10 mg/mL, dilute the VF dye in anhydrous DMSO. Using a glass tube or a vortex, thoroughly mix. Note: It is advised to aliquot the VF storage solution and keep it in the dark at -20°C or -80°C. 3. Determine how much dye working solution is needed. The amount of protein to be labeled determines how much VF dye is needed for the labeling procedure. About 10 is the ideal molar ratio of VF dye to protein. For instance, if 500 μL of 2 mg/mL IgG (MW=150,000) is the necessary labeled protein, and a tube containing 1 mg of VF dye is dissolved in 100 μL of DMSO, 3.95 μL of VF volume is needed. The following is the detailed calculating procedure (with VF 488 as an example): 2) mmol(VF 488) = mmol(IgG)×10 =6.7×10-6 mmol×10 = 6.7×10-5 mmol 3) mmol(IgG) = mg/mL(IgG)×mL(IgG) / MW(IgG) =2 mg/mL×0.5 mL / 150,000 mg/mmol = 6.7×10-6 mmol μL(VF 488) = moles of VF 488The formula for ×MW(VF 488) is 6.7×10-5 mmol / 834 mg/mmol / 0.01 mg/μL = 5.6 μL(VF 488). Method of usage 1. Labeling response 1) Measure out a precise volume of newly made 10 mg/mL VF. The 0.5 mL protein sample solution was gradually mixed with the dye, gently agitated to combine, and then quickly centrifuged to gather the sample at the bottom of the reaction tube. To keep protein samples from becoming denaturated and inactivated, do not mix them vigorously. 2) After gently shaking the reaction vial at room temperature and placing it in a dark area, allow it to incubate for 60 minutes. To improve labeling efficiency, gently invert the reaction vial several times every ten to fifteen minutes to ensure that the two reactants are well mixed. 2. Desalting and purifying proteins An example of a Sephadex G-25 column-based dye-protein conjugate purification procedure is provided below. 1) As directed by the manufacturer, set up the Sephadex G-25 column. 2) Fill the upper part of a Sephadex G-25 column with the reaction mixture. 3) As soon as the sample dips below the top resin's surface, add PBS (pH 7.2–7.4). Remix the necessary sample with extra PBS (pH 7.2–7.4) to finish column purification. Put the parts together that contain the chosen dye-protein combination. Storage temperature: -20°C. Safeguards against storing in the dark. 1. VF dye is light- and humidity-sensitive. Make the VF solution right away, and throw away any leftovers. 2. Minimal amounts of thimerosal (≤0.02 mM or 0.01%) or sodium azide (≤3 mM or 0.02%) won't materially affect protein labeling; nevertheless, 20–50% glycerol will lessen the labeling efficiency. 3. Steer clear of buffers that include ammonium ions or primary amines (such Tris or glycine). They will lessen labeling efficiency by competing with the protein that has to be labeled. 4. This product may not be utilized in food or medication, nor may it be utilized for clinical diagnosis or treatment. It is intended solely for professional scientific research. 5. Please use a lab coat and disposable gloves for your health and safety.

[1]. Nanda JS, et al. Labeling a protein with fluorophores using NHS ester derivitization. Methods Enzymol. 2014;536:87-94.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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 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).

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)]
*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 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
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.)