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.
It is well established that electron-rich ligands such as histidine, tryptophan or cysteine show a relatively high affinity to bond with electropositive transition metals, including Co+2, Ni+2, Cu+2 and Zn+2. This observation has been exploited to improve and control the association of diverse histidine-tagged peptides to liposomes containing metal-chelating lipids. Therefore, immunoliposomes can be generated using nickel-chelating lipids such as Ni-nitrilotriacetic acid (NTA) and 1,2-dioleoyl-sn-glucero-3-[N-(amino-1-carboxypentyl)-iminodiacetic acid) succinyl] (DOGS-NTA aka DGS-NTA), which are commercially available, and His-tagged proteins or peptides. When the liposomes with Ni-NTA headgroups are combined with the His residues, typically at the N- or C-terminus of proteins, the proteins reversibly anchor to the liposomes.
Immunosome®-NTA(Ni) is a non-PEGylated product. For the other reactive (PEGylated and non-PEGyalated products) Immunosome® products suitable for other types of conjugation method see here.
Formulation Information
Immunosome®-NTA(Ni) (Non-PEGylated)
| Lipid Composition | Concentration (mg/ml) | Concentration (mM) | Molar Ratio Percentage |
|---|---|---|---|
| Total | 14.84 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-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) | 0.24 | 0.22 | 1 |
| Buffer and Liposome Size | Specification |
|---|---|
| Buffer | Phosphate Buffered Saline |
| pH | 7.4 |
| Liposome Size | 100 nm |
Conjugation Protocol
Materials and Equipment
In order to conjugate your antibody or protein tagged with FBP to Immunosome®-NTA(Ni) liposomes you will need:
- Laboratory vortex mixer is recommended to have.
- Laboratory magnetic stirrer is needed for dialysis.
- 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 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 total lipid concentration in Immunosome®-NTA(Ni) is 22.45 mM. 1% mol of the lipid in liposomes contains NTA(Ni) 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 the 2 ml volume liposomes, this is equal to 2.20×10-7 mol, and for the 5 ml volume liposomes this is equal to 5.50×10-7 mol of NTA(Ni). Add 1:1 molar ratio of His6-tagged protein, peptide or antibody to NTA(Ni) lipid. Incubate Immunosome®-NTA(Ni) with His6-tagged antibody, protein, peptide or ligand for 1 hour at room temperature.
- Remove the 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 fast, 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. 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.re using either spin column or dialysis cassette. If you do not use the correct MWCO you can lose your entire prep.
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
- Higher concentration of DOGS-Ni-NTA lipid in the liposome formulation will cause an increased change of nonspecific binding and therefore it is important to keep the molar percentage of chelating DOGS-Ni-NTA lipid below 3% to avoid nonspecific binding. Immunosome®-Ni-NTA and Immunodox®Ni-NTA kits all contain 1% DOGS-Ni-NTA which is an optimized concentration for specific binding to His6-tagged proteins, peptides and antibody fragments.
- The binding reaction between a His6-tagged peptide and DOGS-NTA-Ni liposomes will be inhibited in the presence of high concentration of imidazole (~166 mM).
- It should be noted that the use of a 10-histidine tag promoted liposome–liposome crosslinking in the absence of PEG-modified lipids. This could be explained by suggesting that the stretch of 10 histidine residues would be sufficient to bind more than one DOGS-NTA-Ni liposome.
- It has been demonstrated that the presence of PEG polymers inhibits surface reactions between liposomes, and thus can be used to prevent aggregation.
- 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®-NTA(Ni) 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®-NTA(Ni) 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. Faham, Abdus, and Joseph Altin. “Ag‐bearing liposomes engrafted with peptides that interact with CD11c/CD18 induce potent Ag‐specific and antitumor immunity.” International journal of cancer 129, no. 6 (2011): 1391-1403.
2. Faham, Abdus, David Bennett, and Joseph G. Altin. “Liposomal Ag engrafted with peptides of sequence derived from HMGB1 induce potent Ag-specific and anti-tumour immunity.” Vaccine 27, no. 42 (2009): 5846-5854.
3. Bak, Martin, Rasmus I. Jølck, Rasmus Eliasen, and Thomas L. Andresen. “Affinity Induced Surface Functionalization of Liposomes Using Cu-Free Click Chemistry.” Bioconjugate chemistry 27, no. 7 (2016): 1673-1680.
4. Chikh, Ghania G., Wai Ming Li, Marie-Paule Schutze-Redelmeier, Jean-Claude Meunier, and Marcel B. Bally. “Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids.” Biochimica Et Biophysica Acta (BBA)-Biomembranes 1567 (2002): 204-212.
5. Rüger, Ronny, Dafne Müller, Alfred Fahr, and Roland E. Kontermann. “Generation of immunoliposomes using recombinant single-chain Fv fragments bound to Ni-NTA-liposomes.” Journal of drug targeting 13, no. 7 (2005): 399-406.
6. Platt, Virginia, Zhaohua Huang, Limin Cao, Matthew Tiffany, Kareen Riviere, and Francis C. Szoka Jr. “Influence of multivalent nitrilotriacetic acid lipid− ligand affinity on the circulation half-life in mice of a liposome-attached his6-protein.” Bioconjugate chemistry 21, no. 5 (2010): 892-902.
7. Christis, Chantal, and Sean Munro. “The small G protein Arl1 directs the trans-Golgi–specific targeting of the Arf1 exchange factors BIG1 and BIG2.” J Cell Biol (2012): jcb-201107115.
8. Shao, Shuai, Jumin Geng, Hyun Ah Yi, Shobhit Gogia, Sriram Neelamegham, Amy Jacobs, and Jonathan F. Lovell. “Functionalization of cobalt porphyrin–phospholipid bilayers with his-tagged ligands and antigens.” Nature chemistry 7, no. 5 (2015): 438-446.
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
求采纳
3′-Full RACE Core Set Ver.2.0D314 20 次 1,800 元
5′-Full RACE Kit D315 RT反应10次 PCR反应50 次 3,980 元
RACE是基于PCR技术基础上由已知的一段cDNA片段，通过往两端延伸扩增从而获得完整的3'端和5'端的方法
RACE技术的原理和操作方法：http://wenku.baidu.com/link?url=J-9lPSGmRLQriSfXhOqcYYFvJx7ktGIRMgU6k9vDQIywtdJ8deNg7Qi5t4CgtjxuhWUBvQO6_qsULiqUIFckEZty75jYNw5yz6rNqiIKCwm
Clontech的非常好, 感觉. 效果不好可能是RNA问题, 或RTase的问题. 因为目前clontech的RTase是Takara的产品，而非原来clontech的产品，可单购。
步骤：1，RNA抽提（同RT）
2，逆转录，使用合成的oligo
3，p内参
4，使用根据已知序列设计的上游引物和提供下游引物做PCR，注意设计上游引物是退火温度与下游引物相差不大。
5，产物切胶回收，TA克隆测序
这个oligo和下游引物是我根据Takara和BD公司的manual自己设计的，经过我的使用效果很好。具体操作参照takara的3－racemanual。你先做3－race，了解熟悉下，成功后再做5－race。5－race要买试剂盒做，较为复杂！
请具体点，把联系方式和价位都说一下好吗？
cDNA完整序列的获得对基因结构、蛋白质表达、基因功能的研究至关重要。
完整的cDNA 序列可以通过文库的筛选和末端克隆技术获得。
末端克隆技术是20世纪80年代发展起来的。RACE(rapid-amplification of cDNA ends)是通过PCR进行cDNA末端快速克隆的技术。
RACE的优点
与筛库法相比较，有许多方面的优点
1）此方法是通过PCR技术实现的，无须建立cDNA文库，可以在很短的时间内获得有利用价值的信息。
2）节约了实验所花费的经费和时间。
3）只要引物设计正确，在初级产物的基础上可以获得大量的感兴趣基因的全长。
实验室现有的RACE试剂盒的简介
RACE是一种从一个相同的cDNA模板进行5‘和3‘末端快速克隆的方法。此方法会产生较少的错误条带。此过程中使用的酶混合物非常适合长链PCR。
使用此方法的要求是必须知道至少23－28个核苷酸序列信息，以此来设计5’末端和3‘末端RACE反应的基因特异性引物（GSPs）。
RACE引物的设计：
基因特异性引物（GSPs）应该是：
23－28nt
50－70％GC
Tm值≥65度，Tm值≥70度可以获得好的结果
需要实验者根据已有的基因序列设计5‘和3‘RACE反应的基因特异性引物（GSP1和GSP2).由于两个引物的存在，PCR的产物是特异性的。
反应中涉及到的一些事项
cDNA的合成起始于polyA RNA。如果使用其它的基因组DNA或总RNA，背景会很高。
RACE PCR的效率还取决于总的mRNA中目的mRNA的量和不同的引物有不同的退火和延伸温度。
在进行5‘和3’RACE PCR的时候应该使用热启动。
表4中给出了所有引物的相互关系。重叠引物的设计会对全长的产生有帮助。另外，重叠的引物可以为PCR反应提供一个对照。并不是绝对的要利用设计的引物产生重叠片段。
引物GSP中的GC含量要在50－70％之间。这样可以使用降落PCR。避免使用自身互补性的引物序列，否则会产生回折和
如果要用重叠片段来检测设计的引物，GSp1和GSp2之间至少是100－200碱基。只有这样才可以用扩增的产物来鉴定设计的引物是否正确。
降落PCR可以明显的增加RACE PCR产物的特异性。在最开始的循环中，退火温度高于AP1引物的Tm值，可以增加对特异性条带的扩增。随后的退火和延伸的温度降回到AP1的温度，可以进行随后的PCR循环。
形成分子内氢键。另外，避免使用与AP1互补的引物，尤其是在3‘末端。
验证基因特异性引物的对照：
单个引物的阴性对照：只用一个引物GSP来进行阴性对照。这样不应该产生任何的条带。如果可以看到明显的产物，应该改变循环的参数，或重新设计原始引物。
利用两个GSPS进行阳性对照：（只有两个GSP可以产生重叠的时候才可以采用此步。）为了确定RNA样品中目的基因确实表达，利用两个GSP和接头连接的cDNA来产生阳性对照。可以产生两个引物之间的重叠大小的片段。如果没有这个片段，应该重复cDNA的合成，或者从一个不同的组织或细胞来源进行cDNA的合成。
制备和抽提polyA RNA
不要使用DEPC处理过的水。
纯化完mRNA之后，利用琼脂糖凝胶电泳检测mRNA的质量。哺乳动物的mRNA样品是0.5－12kb的拖带，在其中有4.5和1.9kb的rRNA的条带。非哺乳动物的mRNA应略小。
具体的实验步骤
cDNA第一条链的合成：
我们建议进行cDNA合成的对照反应，这样可以对样品的cDNA的合成进行鉴定。加入各种试剂之后，在气浴中42度保温一个小时。
注意： 在水浴或酒精浴中保温回减少反应体积，从而降低第一链的合成效率。
将管放于冰上，以终止第一链的合成反应。
直接进行第二链的合成。
cDNA第二链的合成：
第二链合成的酶混合物中，含有聚合酶、RNaseH和连接酶。T4 DNA聚合酶的功能是补平dscDNA的末端。我们建议做阳性对照，试剂盒中提供人类骨骼肌的mRNA。
建议进行阳性对照,cDNA的质量取决于制备的polyA RNA的质量。非哺乳动物样品的mRNA大约在0.5－3kb之间。
通过电泳检测cDNA的产量，与对照进行对比，这样可以有利于在以后的步骤中对cDNA进行稀释。
接头的连接及连接产物的稀释
按照程序进行连接反应。
如果没有对比样品和对照的产量，利用Tricine－EDTA buffer制备接头连接的ds cDNA的1/50和1/250的稀释物，用两种稀释物进行以下的RACE PCR反应，直到鉴定出哪一种稀释可以得到好的效果。
RACE－PCR扩增
进行5’和3’的RACE－PCR扩增。
利用以下的程序进行降落PCR反应：
注意：
我们建议使用降落PCR反应，这就要求GSP的Tm值≥70度。
当循环结束时，利用1.2%琼脂糖凝胶电泳分析每一个管中的产物5μl，使用适当的分子量marker。
可以根据你的基因的特异性来设计最理想的循环参数。如果看不到带或者只有微弱的带，在68度多加5个循环。最佳的延伸时间取决于扩增条带的长度。如果片断的长度在2－5kb的时候，经常使用4min，0.2-2kb的时候将延伸时间减到2－3min，对于5－10kb的条带，延伸时间增加到10min。
RACE产物的验证：
应该对RACE的片段进行验证,以此来确定是否已经扩增了理想的产物。如果得到的是多条带或者研究的是多基因家族的成员，验证是非常有用的。
有3种验证RACE产物的方法：
（1）比较由GSP和NGSP获得RACE产物。
（2）Southern blot
（3）克隆并测序
我们建议最好测得RACE产物的部分序列。有的时候需要嵌套引物的存在。
比较由GSP和NGSP获得RACE产物
对于5‘末端的RACE产物，比较由AP1和GSP1扩增出来的产物和由AP1和NGSP1扩增出来的产物。
对于3‘末端的RACE产物，比较由AP1和GSP2扩增出来的产物和由AP1和NGSP2扩增出来的产物。这对于鉴定多条带是否是上一个PCR的特异性产物是非常有用的。
如果条带是正确的，在嵌套PCR反应中的条带应该是略微小一些。基本PCR和嵌套PCR产物的迁移率的不同取决于cDNA结构中GSP1和嵌套引物的位置。
RACE产物的克隆和测序：
可以利用胶回收试剂盒来回收RACE产物，此试剂盒适合回收2.5kb以下的RACE产物；对于长的片段，可以通过电洗脱获得好的结果。如果你选择使用其他的纯化方法，最后用Tricine－EDTA buffer 30μl重新悬浮DNA样品。
电泳5μl回收的样品来鉴定回收的质量。
将回收的PCR产物直接克隆到/A型的PCR克隆载体中。另外还可以利用接头和/或cDNA合成引物中的Not1、Srf1、Xma1、ECOR1等酶切位点，将产物克隆到常规载体中.
对于5‘端的RACE产物，我们建议挑取至少8－10个不同的克隆以获得5‘端的最大可能性的序列。（反转录并不总是进行到mRNA模板的5’末端，尤其是长模板。另外，T4 DNA聚合酶会移走5‘末端的0－20个碱基。）
一旦鉴定了含有插入片断的克隆，应该获得多的序列信息。理想的是，可以对整个开放读码框进行测序。包括5‘和3‘的非翻译区。
全长cDNA的获得
通过部分或全部测序鉴定了RACE产物后，可以通过两种选择获得全长的cDNA。
通过PCR的方法获得全长cDNA：
扩增长的cDNA需要较长的延伸时间，但是如果延伸的时间过长，可以产生拖带，所以要慎重的设计引物。
根据从5‘和3‘RACE产物获得序列信息设计5’和3‘GSP引物。这些引物应该来自cDNA的3’或者5‘的末端，应该是23－28nt长。不应该在引物的末端加上限制性位点，这样会导致高背景。在某些时候可以设计3’和5‘的嵌套引物。但是还是应该先利用一对引物进行PCR反应。
进行如下的热循环：
94度 30秒
25个循环 94度 5秒
72度 2－15分钟
延伸的时间应该等于预期的cDNA长度加上2分钟。例如：预期得到6kb的条带，用6＋2＝8分钟的延伸时间。
注意：如果没有条带或者条带弱，增加5个循环；或者优化PCR的条件。
在1.2%的琼脂糖凝胶上分析5μl的样品。通常情况下，可以见到一条单一带，如果这样，利用胶来纯化全长的cDNA。
制备1.2%的TAE buffer制备琼脂糖凝胶。不使用TBE buffer，TBE的胶很难制备全长的cDNA。
将剩下的45μl反应物点样，选用适当的marker。
利用长波紫外观察cDNA（≥300nm）切下全长的cDNA。注意：应该尽量减少紫外对cDNA的照射。
利用胶回收试剂盒回收cDNA。此试剂盒适合回收2.5kb以下的RACE产物；对于长的片段，可以通过电洗脱获得好的结果。如果你选择使用其他的纯化方法，最后用Tricine－EDTA buffer 30μl重新悬浮DNA样品。
将全长的cDNA克隆到T/A型的 PCR克隆载体中。
