WesternBlot時該選擇NC膜還是PVDF膜

1、Western Blot 時該選擇NC膜還是 PVDF 膜？Western Blot時最常用的兩種膜是硝酸纖維素膜(nitrocellulose , NC膜)和 PVDFH (又稱 Positively charged nylon ) ( Polyvinylidene fluoride ,聚偏二氟乙烯膜).這兩種膜各自有什么特點(diǎn)？我們的實驗中該選擇哪一種呢？來看 下面的文字.(摘自?Making and using Antibodies ?)A study of the performance of nitrocellulose, mixed ester, nylon, and covalen

2、t-binding PVDF memberanes after passive protein adsorption and also after electrotransfer was done with several different proteins labeled with125Iodine. Themembranes exhibited different binding capacities in passive adsorption tests with labeled bovine serum albumin. The PVDF showed the least, and

3、the regenerated cellulose and nylon membranes showed the most protein binding. Nitrocellulose and mixed ester membranes were midway between. In tests measuring protein retention, PVDF retained the most bound protein when washed with detergents or 5% skimmed milk. All the membranes showed virtually t

4、he same binding capacity as measured by autoradiography when tested under electrotransfer conditions with Towbin's buffer. In passive adsorption tests, the membranes wxhibited a broad range of capacities but gave similar results in electrotransfer tests.These differences were ascribed to active

5、migration of protein into the membrane matrix instead of simple diffusion and the increased hydrophobicity of Towbin's transfer buffer because of the inclusion of methanol.上面這段文字指出在被動擴(kuò)散轉(zhuǎn)移蛋白時,幾種膜之間結(jié)合蛋白 的水平差異明顯；但是 當(dāng)使用轉(zhuǎn)膜儀轉(zhuǎn)移蛋白時,各種膜之間的差異 就很小了.The choice of membrane used for Western Blot is more criti

6、cal if the blotted protein must maintain its native conformation for detection by the antibody. For example, a comparison using a guanosine triphosphate(GTP)-overlay assay showed that the activity of a bovine GTP-binding protein was barely detectable after transfer to hydrophobic PVDF membranes but

7、was clearly detected after transfer to nitrocellulose. Western blot analysis showed the GTP-binding protein to be present on both PVDF and nitrocellulose membranes, with slightly more detected on the PVDF membranes. The authors speculated that the poor performance of PVDF in the GTP-overlay assay ma

8、y have been due to an inability of GTP-binding protein, thus immobilized, to renature correctly. Therefore, nitrocellulose might be preferred for a Western blot procedure, in which detection requires that the transferred protein regain its native conformation after transfer, such as when the blottin

9、g agent recognized threedimensional structure; for example, an antigenic epitope consisting of noncontiguous residues.由此可見,如果你的抗原表位需要維持其三維結(jié)構(gòu)才能被抗體識 別,就應(yīng)該優(yōu)先選擇NC膜.另外,Abcam網(wǎng)站上技術(shù)資料中的建議是：Two types of membranes are available: nitrocellulose and PVDF. The choice is personal and both work very well. PVDF mem

10、branes require careful pre-treatment: cut the membrane to the appropriate size then soak it in methanol for 1-2 min. Incubate in ice cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold transfer buffer. Failure to do so will cause shrinking while transferring,

11、 and a distorted pattern of transfer.Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich.可見,使用PVDF1時,一定要先用無水甲醇預(yù)處理,再在 transfer buffer中平衡好才可以使

12、用 PVDFM用 甲醇泡的 目 的是為了活化PVD瞋上面的正電基團(tuán),使它更容易跟帶負(fù)電的蛋白質(zhì)結(jié) 合.經(jīng)過預(yù)處理的PVDF1在轉(zhuǎn)膜時,可以使用不含甲醇的 transfer buffer .而使用NC膜時,有的需要用無水甲醇處理,有的 那么不必,直接用transfer buffer平衡好就可以了.注：我使用的 是Pall公司的NC膜,不需要無水甲醇處理,其他公司的不是很清 楚,最好參考產(chǎn)品說明提醒使用PVDF膜的朋友們注意兩點(diǎn)：1. Because of the high number of protein-binding sites in the activated nylon, the ba

13、ckgrouds are normally considerably worse, but careful blocking will eliminate many of these problems.2. Chicken antibodies tend to bind PVDF and other nylonbased membranes, leading to high backgroud. Switching to a nitrocellulose membrane should help reduce background staining.通過上面的分析,根本可以得出結(jié)論,NC膜比P

14、VD瞋更通用一些.盡管NC膜能滿足絕大多數(shù)情況下的要求,大家在使用NC膜時也要注意到NC膜的缺乏之處.Nitrocellulose is the most commonly used, and it or more recently developed derivatives are highly recommended. However, nitrocellulose does have certain disadvantages. The proteins are not covalently bound, and nitrocellulose can be brittle, especi

15、ally when dry. With appropriate care, however, it will fit most applications.下面這段話來自互聯(lián)網(wǎng),僅供參考.硝酸纖維素膜：硝酸纖維素膜是蛋白印跡實驗的標(biāo)準(zhǔn)固相支持 物.在低離子轉(zhuǎn)移緩沖液的環(huán)境下,大多數(shù)帶負(fù)電荷的蛋白質(zhì)會與硝 酸纖維素膜發(fā)生疏水作用而高親和力的結(jié)合在一起,雖然這其中的機(jī)制還不是十分清楚,但由于硝酸纖維素膜的這個特性,而且易于封閉 非特異性結(jié)合,從而得到了廣泛的應(yīng)用.在非離子型的去污劑作用 下,結(jié)合的蛋白還可以被洗脫下來.根據(jù)被轉(zhuǎn)移的蛋白分子量大小, 要選擇不同孔徑的硝酸纖維素膜.由于隨著膜孔徑的不斷減

16、小,膜對 低分子量蛋白的結(jié)合就越牢固.但是膜孔徑如果小于0.1mm蛋白的轉(zhuǎn)移就很難進(jìn)行了.因此,我們通常用0.45仙m和0.2仙m兩種規(guī)格的硝酸纖維素膜.大于20kD的蛋白就可以用0.45 nm的膜,小于 20kD的蛋白就要用0.2仙m的膜了,如果用0.45仙m的膜就會發(fā)生 flowthrough 的現(xiàn)象.從膜的質(zhì)地上來看,最重要的指標(biāo)就是單位面積上能夠結(jié)合的蛋白的量.硝酸纖維素膜的結(jié)合水平主要與膜的 硝酸纖維素的純度有關(guān),市場上有些硝酸纖維素膜通常會還有大量的 醋酸纖維素,因而降低了蛋白的結(jié)合量.如果采用的是100%屯度的硝一 ,一 .一2.酸纖維素,保證了最大的蛋白結(jié)合量,可達(dá) 80-150仙g/cm.由于 100%勺純度,因而也大大減少了非特異性的結(jié)合,降低雜交背景,無 需高嚴(yán)謹(jǐn)度的洗脫步驟.其次,膜的強(qiáng)度和韌性也是需要考慮的因 素.常規(guī)的硝酸纖維素膜比擬脆,漂洗一兩次就會破損,不能反復(fù)使 用.PVD限移膜：PVD呢一種高強(qiáng)度、耐腐蝕的物質(zhì),通常是用來制 造水管的.PVD