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Cy5.5 TEA

CAS No. :

MCE 國(guó)際站：Cy5.5 TEA

產(chǎn)品活性：Cy5.5 TEA 是一種 CY 染料。CY 為花菁 (Cyanine) 的縮寫(xiě)，是由奇數(shù)個(gè)甲基單位連接的兩個(gè)氮原子組成的化合物。菁類(lèi)化合物具有波長(zhǎng)長(zhǎng)、吸收和發(fā)射可調(diào)、消光系數(shù)高、水溶性好、合成相對(duì)簡(jiǎn)單等特點(diǎn)。CY 系類(lèi)染料常被用于蛋白，抗體以及小分子化合物的標(biāo)記，對(duì)于蛋白抗體的標(biāo)記，可以通過(guò)簡(jiǎn)單的混合反應(yīng)來(lái)完成結(jié)合，以下我們介紹了蛋白抗體標(biāo)記的標(biāo)記方法，具有一定的參考意義。

研究領(lǐng)域：Others

作用靶點(diǎn)：Fluorescent Dye

In Vitro: Protocol
1.Protein Preparetion
1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL.
2) The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1 M sodium bicarbonate shall be used for adjustment.
3) If the protein concentration is lower than 2 mg/mL, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL.
4) The protein must be in the buffer without primary amine (such as Tris or glycine) and ammonium ion, otherwise the labeling efficiency will be affected.
2.Dye Preparation (Cy5.5)
Add anhydrous DMSO into the vial of Cy5.5 to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of Cy5.5 required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of Cy5.5 to protein is about 10.
Example: assuming the required marker protein is 500 μL 2 mg/mL IgG (MW=150,000), use 100 μL DMSO dissolve 1 mg Cy5.5, the required Cy5.5 volume is 5.05 μL, and the detailed calculation process is as follows:
1) mmol (IgG) = mg/mL (IgG) ×mL (IgG) / MW (IgG) =2 mg/mL × 0.5 mL / 150,000 mg/mmol= 6.7×10-6 mmol
2) mmol (Cy5.5) = mmol (IgG) × 10 = 6.7×10-6 mmol×10 = 6.7 × 10-5 mmol
3) μL (Cy5.5) = mmol (Cy5.5) ×MW (Cy5.5) / mg/μL (Cy5.5) = 6.7 ×10-5 mmol ×753.88 mg/mmol / 0.01 mg/μL = 5.05 μL (Cy5.5)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL Cy5.5 is slowly added to 0.5 mL protein sample
In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don'tmix well to prevent protein samples from denaturation and inactivation.
2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency.
5.Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a SepHadex G-25 column.
1) Prepare SepHadex G-25 column according to the manufacture instruction.
2) Load the reaction mixture (From "Run conjugation reaction") to the top of the SepHadex G-25 column.
3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

In Vivo: Cy5.5-labeled factor VIIa is developed for imagining cancer. Cy5.5 labeled with these targeting proteins specifically localize to the tumor xenografts for at least 14 days but unconjugated Cy5.5 does not localize to any xenografts or organs. This method of imaging anti-tissue factor in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses. pH/temperature sensitive magnetic nanogels conjugated with Cy5.5-labled lactoferrin (Cy5.5-Lf-MPNA nanogels) are developed as a promising contrast agent for preoperative MRI and intraoperative fluorescence imaging of glioma.

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