Description
Numerous techniques have been developed to prepare immunoliposomes based on the nucleophilic reactivity of free amine groups of proteins or peptides. One of the most popular and commonly used methods is to covalently couple free carboxylic groups to primary amines through activation of the carboxyl groups with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide). EDC, which is a so-called zero-length crosslinking agent, reacts with the carboxyl to form an amine reactive intermediate (O-acylisourea). The produced O-acylisourea can be easily displaced by nucleophilic attack from the primary amino groups in the reaction mixture. However, this intermediate is unstable and hydrolyzed in aqueous solutions. In order to prevent the intermediate hydrolysis, sulfo-NHS (N-hydroxysulfosuccinimide) is added to EDC to produce a significantly more stable and more soluble active intermediate (NHS ester).
Consequently, the immunoliposomes are prepared by a two-step coupling procedure: first, activating the free carboxyl group of the linker lipid incorporated in the liposomes with EDC and sulfo-NHS, and then covalently conjugating the antibodies to the lipids through displacement of sulfo-NHS groups by antibody amines, as depicted below. EDC/sulfo-NHS coupling reactions are highly selective and highly efficient, and the biological activity of the protein or peptide is preserved.
Immunosome®-Succinyl is a non-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®-Succinyl (Non-PEGylated)
Lipid Composition | Concentration (mg/ml) | Concentration (mM) | Molar Ratio Percentage |
---|---|---|---|
Total | 14.79 mg/ml | 22.45 mM | 100 |
L-alpha-Phosphatidylcholine | 12 | 15.5 | 69 |
Cholesterol | 2.6 | 6.73 | 30 |
1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) | 0.19 | 0.22 | 1 |
Buffers and Liposome Size | Specification |
---|---|
Buffer | Phosphate Buffered Saline |
pH | 6 * |
Liposome Size | 100 nm |
* In order to have a highly efficientactivation reaction with EDC and Sulfo-NHS, pH of PBS buffer was adjusted to 6. |
Conjugation Protocol
Materials and Equipment
In order to conjugate the amine on your antibody, protein or peptide to Immunosome®-Succinyl (Non-PEGylated) liposomes you will need:
- EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). The solution should be made fresh moments before use.
- Sulfo-NHS (N-hydroxysulfosuccinimide). The solution should be made fresh moments before use.
- Sephadex® spin column: Sephadex size exclusion spin column can be used for separation of liposomes form free EDC (MW: 191.70). Since EDC is being separated from large liposome particles then any sizes of Sephadex® spin column such as G-10, G-15, G-25, G50 can be used. However, keep in mind that you will lose a large percentage of your liposomes on the spin column. Alternatively, instead of removing the EDC by spin column you can quench it by using 2-mercaptoethanol.
- 2-Mercaptoethanol: To quench the unreacted EDC, 2-mercaptoethanol is added to form a stable complex with the remaining carbodiimide. The 2-mercaptoethanol might not be necessary if you prefer to clean up your liposome from free EDC using a spin column.
- Float-A-Lyzer® with a proper MWCO that easily allows the cleanup of your liposome conjugated ligand from free and non-conjugated protein, peptide or antibody. 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 column or dialysis cassette. If you do not use the correct MWCO, you can lose your entire prep. For this protocol, we recommend MWCO of 300,000 dalton.
Preparation Method
The two-step protocol includes the activation of carboxyl group-containing liposomes with EDC/sulfo-NHS, and subsequent conjugation with the amine group on the proteins, peptides or antibodies:
- In order to activate the carboxyl groups on the liposomes, EDC and sulfo-NHS should be added to the liposomes. The total lipid concentration in Immunosome®-Succinyl (Non-PEGylated) is 22.45 mM. 1% mol of the lipid in liposomes contains COOH 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 2.20×10-7 mol, and for 5 ml volume liposome, this is equal of 5.50×10-7 mol of COOH. Add 10-fold molar excess of EDC and 25-fold molar excess of sulfo-NHS to Immunosome®-Succinyl (Non-PEGylated). To aid in aliquoting the correct amount of these reagents, they may be quickly dissolved in the PBS buffer at a higher concentration, and then a proper volume immediately pipetted into the protein solution to obtain the proper molar quantities.
- Mix well and allow the reaction to proceed for 15 min at room temperature.
- Before adding the protein, peptide or antibody, remove the excess EDC either using a size exclusion spin column, such as Sephadex® spin column or through quenching by 2-mercaptoethanol at a 20 mM final concentration. Addition of 2-mercaptoethanol will not impact the liposomes.
- Dissolve the protein, peptide or antibody at 1-10 mg/ml, depending on the antibody, protein or peptide, in PBS or other amine-free, carboxylate free buffer, pH 7-8.
- Add the protein, peptide or antibody to the EDC/Sulfo-NHS activated Immunosome®-Succinyl (Non-PEGylated) liposomes. The molar ratio of the reactive carboxyl lipid to protein, peptide or antibody is preferred to be around 10:1. The total lipid concentration in our liposomes is 22.45 mM. 1% mol of the lipid in liposomes contains COOH 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 a 2 ml volume liposome this is equal to 2.20×10-7 mol and for 5 ml volume liposomes this is equal of 5.50×10-7 mol of COOH. You will need to calculate the total mol of your peptide, protein or ligand in your solution and add 1:10 molar ratio of ligand to lipid.
- Mix well and allow to react for 2 h at room temperature.
- Remove the non-conjugated protein, peptide or antibody from the immunoliposomes 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; however, 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 will need to choose a cassette with proper MWCO depending on the MW of your protein, peptide, antibody or antibody fragment. NOTE: If you decide to use a dialysis cassette, you will 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 22.45 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
- EDC and sulfo-NHS should be prepared immediately before use and kept at room temperature.
- The activation reaction with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2, and EDC reactions are often performed in at pH 4.7-6.0. For this reason, we have formulated the liposomes in PBS buffer and adjusted the pH to 6.
- Reaction of Sulfo-NHS-activated molecules with primary amines is most efficient at pH 7-8, and Sulfo-NHS-ester reactions are usually performed in phosphate-buffered saline (PBS) at pH 7.2-7.5
- Tris buffer should never be used in any step of the process since it contains amine.
- If you are using a ligand or peptide that is hydrophobic then 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®-Succinyl 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. The liposomes are packaged in an amber 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®-Succinyl 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. Hermanson GT. Bioconjugate techniques. Academic press; 2013 Jul 25.
2. Torchilin V, Weissig V, editors. Liposomes: a practical approach. Oxford University Press; 2003 Jun 5.
3. Grabarek Z, Gergely J. Zero-length crosslinking procedure with the use of active esters. Analytical biochemistry. 1990 Feb 15;185(1):131-5.
4. Yan L, Crayton SH, Thawani JP, Amirshaghaghi A, Tsourkas A, Cheng Z. A pH‐Responsive Drug‐Delivery Platform Based on Glycol Chitosan–Coated Liposomes. Small. 2015 Oct 1;11(37):4870-4.
5. Silva-López EI, Edens LE, Barden AO, Keller DJ, Brozik JA. Conditions for liposome adsorption and bilayer formation on BSA passivated solid supports. Chemistry and physics of lipids. 2014 Oct 31;183:91-9.
6. Hazra M, Singh SK, and Ray S. Surface Modification of Liposomal Vaccines by Peptide Conjugation. Journal of PharmaSciTech, 2011; 1(1): 41-47.
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DXY721认为:
悬浮细胞和贴壁细胞在转染过程中差别不大,主要差别在于转染后的筛选,当然如果你做的是瞬时转染就不存在筛选的问题了。
其实转染的过程很简单,问题是能不能转的进去的,转染率能有多少,转进去是否可以稳定表达目的蛋白等等。
我们也是用脂质体做悬浮细胞的转染,说明书上都有具体的操作过程,将脂质体和目的基因按比例混合,然后加到细胞悬液里就OK了,说的简单,实际上还是有一些细节要注意的,比如脂质体和目的基因混合的比例,转染的细胞数,细胞的代数,细胞的状态,有的还要求在转染的前一天传代一次,不过不要怕,这些在脂质体说明书上都有明确的说明,按照说明书做就可以了。
jinghuanlv认为:
悬浮细胞和贴壁细胞转染还是有很大不同的。
脂质体转染的原理基于电荷吸引原理,先形成脂质体-DNA复合物,散布在细胞周围,然后通过细胞的内吞作用,将目的基因导入细胞内,而脂质体复合物与贴壁细胞的接触机会比悬浮细胞高出很多倍,所以,脂质体转染时悬浮细胞的转染效率要明显低于贴壁细胞。
我们实验室转染悬浮细胞是用的电穿孔法,目前为止,悬浮细胞转染的最好方法还是电转,我们实验室用的电转仪是Bio-Rad的,使用条件是电压250V,电容975uF,效果不错,不妨一用。
本人研究生
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
GFP发出绿色荧光的原理是Ca离子进入GFP的beta-barrel结构中引起的特定能级,因此只要这个结构仍然保持着,就可以发出荧光。
由于GFP的beta-barrel结构非常稳定,一些版本的GFP蛋白(如EGFP)甚至能抵抗94C的高温几分钟而不完全变性,因此想在溶液状态下去掉GFP的荧光是很难的,一般需要用光漂白法。
基于其非常稳定的结构,即便细胞被固定了,仍然会有一部分的GFP蛋白保持其构象而发出荧光。此时荧光可能较弱。在荧光显微镜下是有可能看得到的。
影响转染试验的因素:
1转染试剂跟细胞系不匹配
转染试剂跟细胞系也是讲究配合默契的,使用同一种试剂,不同细胞系转染效率通常不同。但细胞系的选择通常是根据实验的需要,因此在转染实验前应根据实验要求和细胞特性选择适合的转染试剂。每种转染试剂都会提供一些已经成功转染的细胞株列表和文献,通过这些资料可选择最适合实验设计的转染试剂。当然,最适合的是高效、低毒、方便、廉价的转染试剂。
2细胞状态变化
因为有些细胞系是不稳定的,可能随着培养时间的改变,培养条件的不同,不同的选择压力,可能引起不同的克隆选择。因此就算是同一个细胞系,在不同条件下转染能力的差异可能会
很大
(1)转染试剂与细胞不匹配
细胞转染最适合的不是原代细胞,也不是传代很多次的细胞。这是因为细胞培养在实验室中保存数月和数年后会经历突变,总染色体重组或基因调控变化等而演化。这会导致和转染相关的细胞行为的变化。最适合转染的细胞是经过几次传代后达到对数生长期的细胞,细胞生长旺盛,最容易转染。
(2)把握时机
没错!转染也有适当的时机,相比较非分裂细胞——分裂细胞往往要比静止细胞更易于摄取并表达外源DNA。因此对大多数转染操作而言,细胞都在转染当天或前一天种板。
同样重要的是细胞在种板进行转染时不应处于过度生长的状态,如癌细胞数量过多,互相叠加,营养物质耗竭,代谢废物积聚,转染率低下也是很正常的!
因此,一定要在最适细胞密度时转染,才能获得较高的转染率。不同的转染试剂,要求转染时的最适细胞密度各不相同,即使同一种试剂,也会因不同的细胞类型或应用而异。
(3)微生物来捣乱
培养物可被细菌、酵母、真菌、病毒、支原体、甚至其他细胞种类所污染。各种污染都会导致产生错误的结果。
(4)交叉污染
如果同一个实验室同时培养不同种类的细胞,很同意发生“细胞串门”的现象,造成交叉污染。
3转染方法
不同转染试剂有不同的转染方法,但大多大同小异。转染时应跟据具体转染试剂推荐的方法,但也要注意,因不同实验室培养的细胞性质不同,质粒定量差异,操作手法上的差异等,其转染效果可能不同,应根据实验室的具体条件来确定最佳转染条件。
(1)血清
转染后未及时加入血清,会导致细胞大量死亡。一般要在转染后的4-6小时换液且换为有血清的培养基。也可以在原来的无血清培养基里面滴加血清。这个时候,最好不要换液,不要打扰细胞,让它安安静静地休息。但是也不能过早加入血清。过早的话,会引起未转染的细胞疯狂生长。那么,什么是最佳时机呢?在20%的细胞变圆的时候,就是加血清的最佳时机。
不过要特别注意:血清是一种包含生长因子及其它辅助因子的不确切成分的添加物,对不同细胞的生长作用有很大的差别。血清质量的变化直接影响细胞生长,因此也会影响转染效率。新加培养基的预热对细胞转染很有帮助。
(2)DNA质量
DNA质量对转染效率影响非常大。一般的转染技术(如脂质体等)基于电荷吸引原理,如果DNA不纯,如带少量的盐离子,蛋白,代谢物污染都会显著影响转染复合物的有效形成及转染的进行。
4载体构建
转染载体的构建(病毒载体,质粒DNA,RNA,PCR产物,寡核苷酸等)也影响转染结果。因此选择组成或可调控,强度合适的启动子也很重要,同时做空载体及其它基因的相同载体构建的转染正对照可排除毒性影响的干扰。
所以转染用的质粒首先要保证数量,一般为2μg以上。质粒纯度不够或者含有细菌LPS或其他对细胞有毒害作用的物质,也会影响转染效率。这个时候,就应该对质粒进行纯化和浓缩。
以上就是细胞转染率低的主要原因了,在实验过程中有没有遇到什么棘手的问题呢,欢迎留言讨论
做的比较好的,一般都是上海地区的,你可以看下基尔顿生物。