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1、多肽溶解方法和步骤(Peptide dissolving protocol)(2010-05-11 11:19:18)转载田标签：分类：多肽知识多肽 溶解方法 吉尔生化没有绝对理想的一种溶剂可以做到既能溶解所有多肽 ，乂能保持它们的完整性并且与生物学检 测相一致.因此，不得不尝试一系列强溶剂直到多肽溶解.对有溶解困难的多肽，下列方法也许有所帮助.咨询PEPscreen Peptide SetsOption 2Option I11. Ad: 20C uL cf 50% HOA：A'.aie2. Scnoite for 5 m nutesJ1. AJ3 2QQuLc-fDMSC S DWF
2、2. Scnicae for 5 minutes<90% Dissolved1, Add 200 |aL of 50% HOAcin watwr ail peptides2. Sot ote for 5 e niAes>90% Dissolved1. 1* 1M、M ithJ 暨rsc-危edes-2. Scr ate fcr 5 nr nineshsolubleP. Lyccn I ze rrsclubie sect des or peptde2. Dissc vein 1如此5藉。3. ScnicatE fix : minutesR&ptlde SolutionM)rr
3、naliz vckjme ：p cncentrat:rF” working buffer-Reptide Stock Solution*Peptide Storage9第一步总的来说,先用无菌水或者稀醋酸(0.1%)将多肽溶解到一个较高浓度下作为储存溶液，而不是 直接配置到检测浓度.等需要用的时候再把这个储备溶液用缓冲 Buffer配制到需要浓度.比 如一条多肽要溶解成1mg/ml的PBS buffer中，我们需要先将多肽溶解成2mg/ml的储备溶液， 然后在使用前，先移取100ul的10*PBS buffer,再加入400ul的无菌水，最后加入500ul的 2mg/ml储备液.千万不要直接用
4、Buffer缓冲液来溶解多肽，因为很多肽在高盐浓度下溶解度是 下降的,而且如果发生不溶情况，去除这些盐和有机试剂是很困难的一件事.如果多肽溶解过程 中出现可见颗粒始终无法分散到水相中，可以用超声的办法来破碎.不过超声只能加速溶解，并 不能起到改善溶解性的效果.第二步溶解前先审视一下多肽序列，如果多肽中下列氨基酸（Ala，Phe，Ile，Leu，Met，Pro，Val，Trp，Tyr，Cys）占比比较高，这条多肽基本上是难溶的.另外，要注意计算一下序列中含有多少正价基团（Lys，Arg，His和N端）和负价基团（Asp，Glu和C端）， 在中性条件下，最后的净价是正是负？价态为正的，在溶解时可以
5、用稀醋酸调节pH到酸性，价态 为负的，在溶解时可以用稀氨水调节pH到碱性.如果这样仍不能溶解，可以考虑冻十去除溶剂 后变成用强有机溶剂来溶解.第三步如果你的序列在任何pH下都基本不带电荷，或者说你的序列中疏水性氨基酸含量超过50%甚至更高，前面两步基本上是多余的，直接考虑加入少量乙腊，乙醇，DMF或者DMS采溶解，甚至可 以同盐酸月瓜和脉来分散多肽.这些方法溶解多肽的浓度取决于你最终生物检测需要.如果已经 知道一条多肽在水相中溶解不是太好，而最终使用却必须在水相，可以考虑全部用醋酸或者DMF 来溶解，然后缓慢加水来稀释.这样有机溶剂有助于多肽分散到水相中.多肽溶液的储存问题 溶解的多肽的保质期
6、是比较有限的，特别是含有C，M，W，N和Q的多肽.为了延长保存时间，使用 无菌水，保持适当酸性（pH5-6），还有分装冷冻在-20C或者更低是建议采用的方法。另外，一定 要避免反复冻融，这个对多肽的损伤最大。A Strategy for Dissolving Small Sets of PeptidesThe kind of individual treatment described above starts to become impractical whenhandling larger numbers of peptides， say 10 or more. Although exce
7、ptions can be found to the success of any generalised procedure， a recommended strategy for redissolving greater numbers of peptides with varied properties is outlined below.1. Add 0.1% acetic acid/water to give a target peptide concentration of 1-5mg/mL，and sonicate.2. To any insoluble peptides add
8、 pure acetic acid to bring the concentration of acetic acid to 10%(v/v)， and sonicate.3. To any peptides still insoluble add acetonitrile to 20%(v/v)，and sonicate.4. Lyophilise any remaining insoluble peptides to remove the water，acetic acid，andacetonitrile. To the solid， add neat DMFdropwise until
9、the peptide dissolves. Dilute this solution slowly with water to give approximately 10%(v/v) DMF. If the peptide precipitates at any stage during this step, stop adding water and add a little more DMF until the peptide redissolves. Such peptides may be too insoluble in water to be used at concentrat
10、ions equal to the others in the set.5. Dilute each solubilized peptide with the solvent found to be effective for it, to bring the stock solutions to the same peptide concentration. This simplifiescalculations and subsequent handling. Further dilutions, as needed for the bioassay, can be made in the
11、 assay buffer e.g. as a dilution series (titration). Dilution of a relatively insoluble peptide with buffer at this step may successfully avoid precipitation because it is now at a low concentration (below its solubility limit).6. Except after addition of DMF,all solutions can be easily lyophilised
12、to return the peptide to a form suitable for long term storage, if required.This is only one of a large number of possible procedures. The one chosen depends on the assay system, and the need for a particular buffer or peptide concentration.Contact Mimotopes for free technical advice if you wish to
13、use a particular buffer not mentioned here.A Strategy for Dissolving Large Sets of PeptidesPeptide sets such as a General net (Gnet), Replacement net (Rnet) etc. for mass screening are supplied as lyophilised solids in polypropylene tubes. In these cases, the only practical method may be to apply on
14、e solvent to all peptides and use the solutions obtained without trying to optimize for each peptide. For example, for T cell determinant mapping, we have found that a solvent comprising 0.1M HEPES buffer pH7.4 in a 40%acetonitrile/water solution gives effective solutions of most peptides and is non
15、toxic when diluted 20-fold or more in an assay. In this case, sonication of the tubes is also advisable to maximise the dissolution of the peptides. If acceptable in the assay system at the intended working concentration, a good general solvent such as DMFor DMSOould also be used as the first reagen
16、t added to all peptides in the set.On special request, peptide sets can be shipped in the form of frozen solutions, avoiding the problems of redissolving the peptides. Shipping frozen materials adds substantially to the cost of the peptides and can create problems if the peptides are delayed in tran
17、sit.Chemical Changes in Your PeptidesPeptides vary in stability, and a peptide as supplied may soon be degraded if care is not taken to ensure proper storage. In addition to the risk of degradation from proteolytic enzymes, other chemical changes can occur. The short section below is meant to help w
18、ith situations which will commonly arise.1. OxidationA characteristic of cysteine- and methionine-containing peptides is the tendency of these residues to oxidise. Susceptibility to oxidation is sequence-dependent and sometimes even minimal exposure to air of peptides containing these amino acids ca
19、n lead to oxidation. If you wish to avoid oxidation, always work with degassed or deoxygenated solvents and solutions. If possible, maintain peptide solutions at acidic pH (<7). Rate of oxidation increases with pH, so even if the peptide is in the fully reduced form initially, some oxidation will
20、 occur if the peptide is maintained under neutral or basic conditions.Normally, single peptides are consigned in the fully reduced form. If handled properly, they will remain so, but if you need to carry out reduction of a peptide the procedures are as follows:1. Reduction of oxidised cysteine. Diss
21、olve the peptide in 0.1M ammonium bicarbonate containing dithiothreitol (approximately 10-50 fold molar excess) and hold for 4h at room temperature. This procedure is a reasonable starting point butcertain sequences may require more forcing conditions of temperature and time. After reduction, lyophi
22、lise the solution, or de-salt using size exclusion gel chromatography (e.g. Pharmacia Sephadex G-25) or reverse- phase chromatography (e.g.Millipore/Waters Sep-Pak). To prevent re-oxidation, follow the handling procedures mentioned above and store the peptide powder under nitrogen gas and in a freez
23、er.2. Reduction of oxidised methionine by the method of Houghten and Li 2. Dissolve the peptide in 10%acetic acid in water, and add N-methylmercaptoacetamide to 10%(v/v). Incubate at 37 degrees C for 24h or more, then lyophilise or de- salt as for cysteine-containing peptides.2. Other reactionsPepti
24、des have a variety of reactive side chains, and side reactions can occur under both acidic and basic conditions. For example, if glutamine or glutamic acid occurs at the N- terminus of a peptide, cyclisation to form pyroglutamate is likely under acidic conditions (10% acetic acid). Mild basic soluti
25、ons (0.1M ammoniunbicarbonate) will lead to imide formation in asparagine-containing peptides. Whendissolving single peptides, avoid conditions known to promote side reactions with the residues present.Degradation due to microbial growth should not occur provided sterile distilled water or buffers a
26、re used, and solutions are frozen for storage. Sterilizing filtration of the peptide solution is another option, which also removes traces of insoluble or particulate materials. If filtration is chosen, ensure that the filters used are resistant to the solvent in which the peptide is dissovled, and have low peptide-binding properties.