Description
During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus or the available sulfur (e.g. Fab’ fraction or thiolated antibodies). Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge. Coupling of sulfhydryl groups with maleimide groups has been the most widely used conjugation of antibodies to liposomes. Different lipids which are offered for thioether conjugation contain maleimide, aromatic maleimides such as N-[4-(p-maleimidophenyl)-butyryl] (MPB) or 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC) group. The maleimide function group of MCC which contains an aliphatic cyclohexane ring is more stable toward hydrolysis in aqueous reaction environments rather than the aromatic phenyl group of MPB. MPB and MCC lipids are non-PEGylated lipids and they have separate kits and protocols than PEGylated maleimide lipids.
One of the major problems of using maleimide chemistry for conjugation is the rapid hydrolysis of maleimide lipid. The rate of hydrolysis is much faster in alkaline pH and therefore, controlling the pH throughout the entire process is necessary and it is recommended to use the pH of 7. Due to the hydrolysis of maleimide group, our kits are designed for post-insertion of ligand conjugated maleimide lipid into the preformed liposomes. After post conjugation, the liposomes must be used right away because hydrolysis may occur after sulfhydryl coupling to the maleimide as well. Another problem is the reactivity and oxygen sensitivity of sulfhydryl group on thiolated antibody or Fab’ fragment. Due to that the conjugation reaction should be done under argon or nitrogen using inflatable polyethylene glove bag chambers.
Thiolation, which is adapted to the modification of all the antibody functional groups, is relatively clean, fast, and efficient. However, different antibodies may be more sensitive to some procedures than others. Therefore, it is recommended to select the chemistry and site of modification depending on what procedures are compatible with the antibody.
Immunosome®-Maleimide is a PEGylated product. For the other sulfhydryl reactive (PEGylated and non-PEGyalated products) and also Immunosome® products suitable for other types conjugation methods see here.
Formulation Information
Immunosome®-Maleimide (PEGylated) (Post-insertion)
Post-insertion Kit (3 Vials) | Specification |
---|---|
Vial 1 | Preformed liposomes composed of HSPC and Cholesterol (60:40 molar ratio) |
Vial 2 | DSPE-PEG(2000)-Maleimide lipid (reactive PEGylated lipid) in powder form |
Vial 3 | DSPE-PEG(2000) lipid (non-reactive PEGylated lipid) in powder form |
Lipid Composition for Vial 1* | Concentration (mg/ml) | Concentration (mM) | Molar Ratio Percentage |
---|---|---|---|
Hydrogenated Soy PC | 11.5 | 14.66 | 60 |
Cholesterol | 3.83 | 9.9 | 40 |
Total | 15.33 mg/ml | 24.56 mM | 100 |
* For the 5-ml kit, the volume of vial 1 is 4 ml. 1 ml of micelle solution that are formed using vials 2 and 3 will be added to this vial to make the final volume of 5 ml in the final product. For the 2-ml kit, the volume of vial 1 is 1.6 ml. 0.4 ml of micelle solution that is formed using vials 2 and 3 will be added to this vial to make the final volume of 2 ml in the final product. |
Buffer and Liposome Size for Vial 1 | Specification |
---|---|
Buffer | Phosphate Buffered Saline |
pH | 7.4 |
Liposome Size | 100 nm |
Vial 2 * | Specification |
---|---|
DSPE-PEG(2000)-Maleimide Lipid | This vial contains reactive DSPE-PEG(2000)-Maleimide lipid in powder form. This lipid is conjugated to a reactive protein, peptide or ligand containing sulfhydryl and then mixed with non-reactive DSPE-PEG(2000) lipid in aqueous solution to form micelles. The PEGylated lipid micelles are incubated with preformed liposomes in vial 1 and PEG lipids will post-insert themselves into the liposomes. |
* The amount of the powdered PEG(2000)-Maleimide lipid for 2-ml kit is 1.34 mg and for 5-ml kit is 3.34 mg. |
Vial 3 * | Specification |
---|---|
DSPE-PEG(2000) Lipid | This vial contains non-reactive DSPE-PEG(2000) lipid in powder form. This lipid in mixed with DSPE-PEG(2000)-NHS lipid which is already conjugated to a ligand (protein, peptide, etc.) in aqueous solution to form micelles. The PEGylated lipid micelles are incubated with preformed liposomes in vial 1 and PEG lipids will post-insert themselves into the liposomes. |
* The amount of the powdered PEG(2000)-DSPE lipid for the 2-ml kit is 5 mg and for the 5-ml kit is 12.5 mg. |
Conjugation Protocol (Post-insertion)
Materials and Equipment
The 3-vial post-insertion kit contains preformed liposomes (vial 1), DSPE-PEG(2000)-Maleimide lipid in powder form (vial 2) and non-reactive PEGylated lipid in powder form (vial 3). In order to use the post-insertion kit, you will need:
- Two small 10-ml round bottom flasks or two small glass vials.
- A rotary evaporator. We understand that many labs might not have a rotovap. Alternatively, you can use a nitrogen tank connected to a thin hose for creating a stream of nitrogen flow to dry the lipid and make a thin film.
- A small amount of a solvent such a chloroform or methylene chloride (you will only need a few milliliters).
- Phosphate buffered saline (PBS). pH should be adjusted to 7.
- 2-mercaptoethanol.
- Aldrich®-Atmosbag connected to a nitrogen tank. Due to oxygen sensitivity of the reaction, the coupling reacting should be done in oxygen-free environment.
- 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. For this protocol, we recommend MWCO of 300,000 dalton.
- A Sonicator. It is better to have a bath sonicator. If you do not, that is fine, and you still can follow the protocol. You may also use a vortex instead of the sonicator for agitation of the solution as well.
Preparation Method
- The post-insertion kits come in two sizes; 2 ml and 5 ml. For the 2-ml kit size, dissolve the content of vial 3 (non-reactive PEGylated lipid) in 100 µl of chloroform or methylene chloride. For the 5-ml kit size, the content of vial 3 should be dissolved in 250 µl of chloroform or methylene chloride. Transfer the solution to a 10-ml round bottom flask. Dry the chloroform using a rotary evaporator or under a stream of nitrogen in order to make a dried lipid film.
- For the 2-ml kit, add 100 µl of PBS buffer to the dried lipid film. For the 5-ml kit, the amount of the added buffer is 250 µl. It is preferred to sonicate the hydrated lipid film using a bath sonicator and sonicate the micelle solution for 5 minutes. If you do not have a bath sonicator then hydrate the dried lipid film with PBS for at least 1 hour and constantly rotate the solution in the round bottom flask using a rotavap (not connected to vacuum) or by hand to make sure that all the dried lipid on the wall of the round bottom flask will go to the solution and form micelles. Alternatively, you can use a vortex to agitate the solution. The goal is to have all the dried lipid on the wall of the round bottom glass to go to the micelle solution. Cover the mouth of the round bottom flask with parafilm. Refrigerate the micelle solution of non-reactive PEG lipids until it is ready to be mixed with micelles formed in the step 5.
- The 2-ml kit contains 1.30 mg (0.22 µmol) of reactive DSPE-PEG(2000)-Maleimide lipid (vial 2). The 5-ml kit contains 3.25 mg (0.55 µmol) of reactive DSPE-PEG(2000)-Maleimide lipid (vial 2). For the 5-ml kit size, the content of vial 2 (DSPE-PEG(2000)-Maleimide lipid) should be dissolved in 250 µl of chloroform or methylene chloride. Transfer the solution to a 10 ml round bottom flask. Dry the chloroform using a rotary evaporator or under a stream of nitrogen to make a dried lipid film.
- Dried DSPE-PEG-Maleimide film is hydrated with PBS buffer to form a micellar lipid solution. If you are using the 2-ml post-insertion kit, then hydrate the 1.30 mg of dried DSPE-PEG-Maleimide lipid film in 100 µl of buffer, and if you are using the 5-ml post-insertion kit, then hydrate the 3.25 mg of dried DSPE-PEG-Maleimide lipid film in 250 ml of buffer.
- Incubate the micellar lipid solution with the antibody, protein or peptide at 3:1 molar ratio or lipid to protein. Allow the reaction to proceed in phosphate buffer under the nitrogen (inert gas) chamber for 8 hours at room temperature with moderate stirring. The concentration of antibody, peptide or protein that is added to micellar solution is depend on the solubility of your molecule. It is recommended to use a fairly concentrated solution. For example, use a volume around 100 µl of antibody, peptide or protein for 2-ml kit and around 250 µl of antibody, peptide or protein for 5-ml kit.
- The excess maleimide groups were capped by reaction with 2-mercaptoethanol. The reaction is quenched with 2 mM 2-mercaptoethanol for 30 min.
- The micelles obtained from the steps 2 and 5 are mixed. Total volume of the 2 mixed micelles for the 2-ml kit is 300 µl and for the 5-ml kit is 750 µl. Incubate the mixed micelles with preformed liposomes (vial 1) at 60℃ for 30 min.
- 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 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.
Quantification of reactive sulfhydryl in antibodies or ligands (Ellman’s Assay)
The yield of conjugation is the most important factor in formulating immunoliposomes. Many scientists simply assume that their thiolated antibody or the Fab’ fraction contains reactive sulfhydryl for conjugation to maleimide lipid without further assaying. Disulfide bridge can form very easily so it is very important to quantify the available reactive sulfhydryl in your antibody or ligand solution before performing the conjugation reaction with maleimide liposomes.
Ellman’s assay is a widely used assay for determining the amount of free sulfhydryl. You can follow the step by step protocol here.
Liposome Particle Calculator
Immunosomes are unilamellar liposomes and sized to 100 nm. The molar concentration of liposome is 24.56 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
- After conjugation reactions, liposomes containing excess maleimide or thiol groups may exhibit undesirable qualities, such as aggregation, reactions in vitro and in vivo, and immunogenicity. These reactive moieties can be quenched with reagents containing iodo-, maleimide, or sulfhydryl groups where appropriate. This is likely to be a particularly serious problem for thiolated liposomes. Therefore, it is recommended that the antibody be thiolated to generate the appropriate reactive entities for the final conjugation reaction.
- In order to prevent oxidation of sulfhydryl on antibody and formation of disulfide bridge, the coupling reaction must be performed under an inert atmosphere such as argon or nitrogen. To set up an inert gas chamber we recommend using Aldrich®-Atmosbag with is a flexible, inflatable polyethylene chamber with built-in gloves which is a portable and inexpensive alternative to laboratory glove box.
- Maleimide group on lipid is highly sensitive of alkaline pH and it will hydrolyze rapidly at higher pH. Experimental investigations have been shown that in alkaline condition (pH > 7.5), maleimide and its derivatives are hydrolyzed to a non-reactive maleamic acid (see the figure below). This instability should be considered in any quantitative procedures, such as coupling with sulfhydryl groups. Therefore, it is very important to make sure that the pH of the reaction with stay between 6.5 and 7 during the entire process.
- 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®-Maleimide (PEGylated) post-insertion kit comes in three vials: vial 1 is a white translucent liquid made of nano size unilamellar liposomes which does not contain any reactive of non-reactive PEGylated lipid. Usually due to the small size of liposomes no settling will occur in the bottom of the vial. Vial 2 contains reactive DSPE-PEG(2000)-Maleimide lipid in white powder form. Vial 3 contains non-reactive DSPE-PEG(2000) lipid in white powder form.
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®-Maleimide kit 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. Matsui, S., and H. Aida. “Hydrolysis of some N-alkylmaleimides.” Journal of the Chemical Society, Perkin Transactions 2 12 (1978): 1277-1280.
2. Barradas, Remigio Germano, Stephen Fletcher, and John Douglas Porter. “The hydrolysis of maleimide in alkaline solution.” Canadian Journal of Chemistry 54.9 (1976): 1400-1404.
3. Gregory, John D. “The stability of N-ethylmaleimide and its reaction with sulfhydryl groups.” Journal of the American Chemical Society 77.14 (1955): 3922-3923.
4. Nassander UK, Steerenberg PA, De Jong WH, Van Overveld WO, Te Boekhorst CM, Poels LG, Jap PH, Storm G. Design of immunoliposomes directed against human ovarian carcinoma. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1995 Apr 12;1235(1):126-39.
5. Derksen JT, Morselt HW, Scherphof GL. Uptake and processing of immunoglobulin-coated liposomes by subpopulations of rat liver macrophages. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1988 Sep 16;971(2):127-36.
6. Derksen JT, Scherphof GL. An improved method for the covalent coupling of proteins to liposomes. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1985 Mar 28;814(1):151-5.
7. Nässander UK, Steerenberg PA, Poppe H, Storm G, Poels LG, De Jong WH, Crommelin DJ. In vivo targeting of OV-TL 3 immunoliposomes to ascitic ovarian carcinoma cells (OVCAR-3) in athymic nude mice. Cancer research. 1992 Feb 1;52(3):646-53.
8. Park JW, Hong K, Carter P, Asgari H, Guo LY, Keller GA, Wirth C, Shalaby R, Kotts C, Wood WI. Development of anti-p185HER2 immunoliposomes for cancer therapy. Proceedings of the National Academy of Sciences. 1995 Feb 28;92(5):1327-31.
9. Koning GA, Morselt HW, Velinova MJ, Donga J, Gorter A, Allen TM, Zalipsky S, Kamps JA, Scherphof GL. Selective transfer of a lipophilic prodrug of 5-fluorodeoxyuridine from immunoliposomes to colon cancer cells. Biochimica et Biophysica Acta (BBA)-Biomembranes. 1999 Aug 20;1420(1):153-67.
10. Vingerhoeds MH, Steerenberg PA, Hendriks JJ, Dekker LC, Van Hoesel QG, Crommelin DJ, Storm G. Immunoliposome-mediated targeting of doxorubicin to human ovarian carcinoma in vitro and in vivo. British journal of cancer. 1996 Oct 1;74(7):1023-9.
11. Kirpotin D, Park JW, Hong K, Zalipsky S, Li WL, Carter P, Benz CC, Papahadjopoulos D. Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro. Biochemistry. 1997 Jan 7;36(1):66-75.
12. Garnier B, Bouter A, Gounou C, Petry KG, Brisson AR. Annexin A5-functionalized liposomes for targeting phosphatidylserine-exposing membranes. Bioconjugate chemistry. 2009 Oct 19;20(11):2114-22.
13. Mattson G, Conklin E, Desai S, Nielander G, Savage MD, Morgensen S. A practical approach to crosslinking. Molecular biology reports. 1993 Apr 1;17(3):167-83.
14. Smyth DG, Blumenfeld OO, Konigsberg W. Reactions of N-ethylmaleimide with peptides and amino acids. Biochemical Journal. 1964 Jun;91(3):589-95.
15. Harokopakis E, Childers NK, Michalek SM, Zhang SS, Tomasi M. Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens. Journal of immunological methods. 1995 Sep 11;185(1):31-42.
16. Gradauer K, Vonach C, Leitinger G, Kolb D, Fröhlich E, Roblegg E, Bernkop-Schnürch A, Prassl R. Chemical coupling of thiolated chitosan to preformed liposomes improves mucoadhesive properties. International journal of nanomedicine. 2012;7:2523-34.
17. Gradauer K, Barthelmes J, Vonach C, Almer G, Mangge H, Teubl B, Roblegg E, Dünnhaupt S, Fröhlich E, Bernkop-Schnürch A, Prassl R. Liposomes coated with thiolated chitosan enhance oral peptide delivery to rats. Journal of controlled release. 2013 Dec 28;172(3):872-8.
18. Moreira JN, Ishida T, Gaspar R, Allen TM. Use of the post-insertion technique to insert peptide ligands into pre-formed stealth liposomes with retention of binding activity and cytotoxicity. Pharmaceutical research. 2002 Mar 1;19(3):265-9.
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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
脂质体介导法
实验原理
上图所示是脂质体介导转染的示意图,它显示了外源质粒进入细胞的一般过程。
外源基因进入细胞主要有四种方法:电击法、磷酸钙法和脂质体介导法和病毒介导法。电击法是在细胞上短时间暂时性的穿孔让外源质粒进入;磷酸钙法和脂质体法是利用不同的载体物质携带质粒通过直接穿膜或者膜融合的方法使得外源基因进入细胞;病毒法是利用包装了外源基因的病毒感染细胞的方法使得其进入细胞。但是由于电击法和磷酸钙法的实验条件控制较严、难度较大;病毒法的前期准备较复杂、而且可能对于细胞有较大影响;所以现在对于很多普通细胞系,一般的瞬时转染方法多采用脂质体法。
利用脂质体转染法最重要的就是防止其毒性,因此脂质体与质粒的比例,细胞密度以及转染的时间长短和培养基中血清的含量都是影响转染效率的重要问题,通过实验摸索的合适转染条件对于效率的提高有巨大的作用。
上图是本次实验采用的脂质体中阳离子组分的结构的示意图。
本次实验采用的脂质体是promega公司的TransFast脂质体试剂,它是一种阳离子脂质体和中性脂质体的混合物,是对于本次实验中采用的293T细胞优化的转染试剂。
实验室一直都是用日常型质粒抽提试剂盒,转染细胞没问题。
我觉得只要是注意以下2点就可以了:
1,注意大肠杆菌(Escherichia coli)本身的污染,收集菌体沉淀时防止菌液散落,经常用75%的乙醇擦拭手套。
2,最后洗脱时最好使用无内毒素的水,我们是用注射用水的。
无论是小提还是大提我们都是用的日常型的,并没有刻意用转染级的,因为转染量大,去内毒素的操作太麻烦,损失太大。
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.
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。
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