Description
In order to develop a rapid and straightforward coupling procedure at the PEG terminus, a method of direct coupling antibodies to the PEG terminus of liposomes was introduced by Bendas et al. [1]. In this methodology, antibodies are simply attached to the PEG terminus of liposomes, which had been end-group functionalized with cyanuric chloride, in mild basic conditions (pH 8.8) without prior antibody derivatizations. It has been shown that in order to obtain a stable attachment of proteins on liposome, the DSPE-PEG-cyanur was added into the liposomes to chemically conjugate with proteins to form a stable complex and minimize the denaturation of proteins.
Proteins can be covalently coupled to the liposomes via amine-reactive cyanur-groups, either directly to the vesicle surface using cyanuric chloride-activated DSPE (cyanur-DSPE) or to the distal ends of PEG-spacers using activated cyanur-PEG-PE (ammonium salt). Cyanuric chloride at the PEG terminus functions to link peptides, antibodies and other amine-containing biomolecules or nanoparticles via a nucleophilic substitution reaction under basic conditions. Antibodies or other proteins can be conjugated without any previous derivatization.
Immunosome®-Cyanur is a PEGylated product. For other amine reactive (PEGylated and non-PEGyalated products) and also Immunosome® products suitable for other types conjugation methods see here.
Formulation Information
Immunosome®-Cyanur (PEGylated)
Lipid Composition | Concentration (mg/ml) | Concentration (mM) | Molar Ratio Percentage |
---|---|---|---|
Total | 15.92 mg/ml | 21.58 mM | 100 |
Hydrogenated Soy PC | 9.58 | 12.22 | 57 |
Cholesterol | 3.19 | 8.25 | 38 |
DSPE-PEG(2000) | 2.5 | 0.89 | 4 |
DSPE-PEG(2000)-Cyanur | 0.65 | 0.22 | 1 |
Buffer and Liposome Size | Specification |
---|---|
Buffer | Borate Buffer |
pH | 8.8 |
Liposome Size | 100 nm |
Conjugation Protocol
Materials and Equipment
In order to conjugate your antibody, protein, peptide or ligand to Immunosome®-Cyanur (PEGylated) you will need:
- Float-A-Lyzer® with a proper MWCO that easily allows the cleanup of your liposome conjugated ligand from free and non-conjugated protein/peptide/ligand. You need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton the pore size on the dialysis membrane gets close to 100 nm and therefore your liposomes can be dialyzed out. You cannot use dialysis cassettes blindly. Please understand the technique before using either spin columns or dialysis cassettes. If you do not use the correct MWCO you can lose your entire prep. In this case, we recommend using a dialysis cassette with MWCO of 300,000 dalton.
- Borate buffer. You can either make the borate buffer or purchase it from a chemical vendor. In any case, you need to make sure that the pH is adjusted to 8.8.
Preparation Method
- The total lipid concentration in Immunosome®-Cyanur is 21.58 mM. 1% mol of the lipid in liposomes contains PEG-Cyanur group and only half of them are exposed to the outside of the liposomes, which is equal to 0.11 mM of reactive conjugable lipid. For 2 ml volume liposome, this is equal to 2.2×10-7 mol, and for 5 ml volume liposome, this is equal to 5.5×10-7 mol of PEG-Cyanur.
- Add 1:1000 molar ratio of antibody, protein, peptide or ligand to total lipid. This will be equal to 1:5 molar ratio of antibody, protein, peptide or ligand to PEG-Cyanur lipid. For example, in a 2-ml kit, for 2.2×10-7 mol of PEG-Cyanur lipid, 4.4×10-8 mol of antibody, protein, peptide or ligand is needed.
- The conjugation must be done under mild basic condition such a borate buffer pH 8.8. Dissolve your antibody, protein, peptide or ligand in borate buffer with pH 8.8.
- Incubate Immunosome®-Cynaur with antibody, protein, peptide or ligand for 16 hours at room temperature.
- Remove non-conjugated antibody, protein, peptide or ligand by dialysis. We prefer dialysis to size exclusion columns. Dialysis is a much slower process but there will be minimum loss of immunoliposomes after the prep is cleaned from non-conjugated protein/peptide/ligand. Spin columns are much faster, but you can easily lose over 50% of the liposomes on the spin column. We recommend using Float-A-Lyzer® dialysis cassette from Spectrum Labs. You need to choose a cassette with proper MWCO depending on the MW of your protein, ligand, antibody or antibody fragment. In this case we recommend using a dialysis cassette with MWCO of 300,000 dalton. NOTE: If you decide to use a dialysis cassette, you need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton the pore size on the dialysis membrane gets close to 100 nm and therefore your liposomes can be dialyzed out. You cannot use dialysis cassettes and spin columns blindly. They come in various sizes and you need to choose the correct size wisely. Dialyze the immunoliposome solution in 1 liter of PBS at pH 7.4 for 8 hours. Change the dialysis buffer with a fresh 1 liter of PBS and let is dialyze for another 8 hours. After this step, your cleaned up immunoliposome is ready to be used.
Liposome Particle Calculator
Immunosomes are unilamellar liposomes and sized to 100 nm. The molar concentration of liposome is 21.58 mM. By having liposome diameter (nm) and lipid concentration (µM), you can calculate the total number of the lipids in one liposome and the number of the liposomes in one milliliter of the liposome solution. To use the calculator click here.
Technical Notes
- Tris buffer should never be used in any step of the process since it contains amine.
- Cyanuric chloride is considered as a sensory respiratory irritant. However, despite the name of the cyanur-modified liposomes, they have not shown any sign of acute, chronic or genotoxicity.
- Cyanur groups are amine-reactive, however, some random attachments of the antibodies can be expected since cyanuric chloride can react with a wide range of nucleophilic functionalities, such as alcohols and thiols. This may interfere with the binding of the antibody to the liposome, and therefore, the binding affinity would change.
- Size exclusion spin columns such as Sepharose® CL-4B can be used instead of Float-A-Lyzer® dialysis cassette. However, a very large amount of liposomes will stick to the column during the cleanup process and therefore we strongly suggest using dialysis than size exclusive beads.
- If you are using a ligand or peptide that is hydrophobic, it is recommended to solubilize it in DMSO or DMF and then add the buffer to it. It is recommended not to use more than 5% volume of DMSO or DMF in the solution. DMF and DMSO are both compatible with liposomes and they are also miscible in water. Other organic solvent such as ethanol and chloroform are not compatible with liposomes and will cause the liposomes to lyse. If you end up using DMSO or DMF then after the conjugation reaction is done, you need to remove DMSO and DMF from the liposomes. In order to do that you need to use a dialysis cassette that is made from REGENERATED CELLULOSE MEMBRANE. NOTE: Not all membranes are compatible with DMF and DMSO. We recommend using a Slide-A-Lyzer™ MINI Dialysis Device with MWCO of 2K made from regenerated cellulose membrane manufactured by ThermoFisher. After DMSO or DMF is removed, you can use Float-A-Lyzer® dialysis device for the final step of cleaning up the prep.
- Liposomes should be kept at 4°C and NEVER be frozen.
Database
Direct link to the database page for easy navigation: Immunoliposomes Conjugation Database
Appearance
Immunosome®-Cyanur is a white translucent liquid made of nano size unilamellar liposomes. Usually due to the small size of liposomes no settling will occur in the bottom of the vial.
Ordering/Shipping Information
- All liposome based formulations are shipped on blue ice at 4°C in insulated packages using overnight shipping or international express shipping.
- Liposomes should NEVER be frozen. Ice crystals that form in the lipid membrane can rupture the membrane, change the size of the liposomes and cause the encapsulated drug to leak out. Liposomes in liquid form should always be kept in the refrigerator.
- Clients who order from outside of the United States of America are responsible for their government import taxes and customs paperwork. Encapsula NanoSciences is NOT responsible for importation fees to countries outside of the United States of America.
- We strongly encourage the clients in Japan, Korea, Taiwan and China to order via a distributor. Tough customs clearance regulations in these countries will cause delay in custom clearance of these perishable formulations if ordered directly through us. Distributors can easily clear the packages from customs. To see the list of the distributors click here.
- Clients ordering from universities and research institutes in Australia should keep in mind that the liposome formulations are made from synthetic material and the formulations do not require a “permit to import quarantine material”. Liposomes are NOT biological products.
- If you would like your institute’s FedEx or DHL account to be charged for shipping, then please provide the account number at the time of ordering.
- Encapsula NanoSciences has no control over delays due to inclement weather or customs clearance delays. You will receive a FedEx or DHL tracking number once your order is confirmed. Contact FedEx or DHL in advance and make sure that the paperwork for customs is done on time. All subsequent shipping inquiries should be directed to Federal Express or DHL.
Storage and Shelf Life
Storage
Immunosome® products should always be stored at in the dark at 4°C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. If the suspension is frozen, the encapsulated drug can be released from the liposomes thus limiting its effectiveness. In addition, the size of the liposomes will also change upon freezing and thawing.
Shelf Life
Immunosome®-Cyanur is made on daily basis. The batch that is shipped is manufactured on the same day. It is advised to use the products within 4 months of the manufacturing date.
References and background reading
1. Bendas G, Krause A, Bakowsky U, Vogel J, Rothe U. Targetability of novel immunoliposomes prepared by a new antibody conjugation technique. International journal of pharmaceutics. 1999 Apr 20;181(1):79-93.
2. Lee HY, Mohammed KA, Kaye F, Sharma P, Moudgil BM, Clapp WL, Nasreen N. Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer. International journal of nanomedicine. 2013;8:4481.
3. Nyanhongo GS, Steiner W, Gübitz GM, editors. Biofunctionalization of Polymers and their Applications. Springer Science & Business Media; 2011 Aug 6.
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大家都是用什么方法挑选细胞单克隆的
单克隆:单克隆是指‘子代来源于一个母体.
细胞培养:细胞的大规模克隆.细胞培养,既包括微生物细胞的培养,细胞培养技术可以由一个细胞经过大量培养成为简单的单细胞或极少分化的多细胞.
单克隆一般常指动植物细胞的克隆,细胞培养一般是指动物、微生物等细胞的细胞克隆.
二者没有什么明显区别.单克隆在单克隆抗体制备中比较常见.其实是对骨髓瘤细胞的细胞培养.
目标蛋白对细胞有毒性,导致细胞死亡;
转染试剂以及DNA用量信息需要优化,否则对细胞具有伤害;
细胞贴壁转染之后没有正常换液。
建议:考虑对目标蛋白进行截短构建、尝试其他细胞系统;摸索转染试剂以及DNA用量信息,如果转染试剂毒性太大,可以考虑尝试义翘转染试剂sinofection;对转染后的细胞进行换液处理,如果细胞状态感觉不够理想,可以考虑添加一些血清来帮助细胞恢复健康。
以上所有分析、建议的前提是,细胞培养、无菌操作等等都没有问题。祝顺利,加油~
我转的是7901、7901/DDP两种细胞,前者7901细胞很容易就转上,并且转后,状态良好,可是7901/DDP一转就死,我用的是吉玛慢病毒,转24小时后换液,刚开始一两天,没有异常,但后来细胞慢慢就死了,并且不是漂浮的,很多是贴着壁死,像是瓦解了一样
这是未转时细胞的样子
这是细胞转后,死亡的样子
并且即使是有些细胞未死,细胞后来也变得很脏,感觉有很破碎的细胞碎片
本人实验小白,**园子里大神指点,急,实在不知道怎么回事
“转化”是指含外源基因的重组质粒(载体)将外源基因直接导入原核细胞(如细菌);
“转导”指通过重组病毒载体将外源基因导入真核细胞或原核细胞;
“转染”指重组质粒载体或游离核苷酸在脂质体等介导下进入真核细胞;
“感染”在基因转移实验中强调重组病毒载体入侵受体细胞的过程。
在使用这4 个名词时, 应仔细分析基因转移实验的四要素——转移物、载体、介导方法、受体细胞类型,而正确区 分载体和受体细胞类型是辨析的关键点。当载体是重组质粒时,如受体细胞是原核细胞应使 用“转化”,如受体细胞是真核细胞则使用“转染”;当载体是重组病毒时,如强调转移物进 入受体细胞应使用“转导”,如强调重组病毒载体进入受体细胞的过程则使用“感染”。
各位版友求助,
我使用Hek293构建转染模型,瞬转5质粒,用lipo2000做转染体系。
转染48h,发现荧光较强的细胞都在爬片的边缘,比例十分少。是因为我添加试剂的手法不对吗。
同时也发现加入转染体系后细胞状态特别差。想问一下用lipo2000时可以用无双抗的10%FBSDMEM吗。我转染前6h现在用的是纯DMEM,不含FBS。
请各位大神帮忙
转染分2种,一种是瞬时转染,即转染后让细胞表达目的蛋白后即提取蛋白,提一次蛋白,转染一次,这种方式一般不传代;
另一种转染为稳定转染,转染后加入一定选择压力进行筛选,没有转染的细胞不能存活,只留下转染的细胞,这种情况下可以筛选单个转染细胞,构建稳定表达某一特定蛋白或基因的细胞系。
其次要看下你选择单位的规模如何,做的比较好的,还是上海这边的,你可以看下基尔顿生物,原代细胞培养,动物造模,整体课题外包。
暂无品牌问答